Methods and compositions for universal detection of nucleic acids

ABSTRACT

Provided are methods and compositions for detecting the presence or amount of one or more target nucleic acids in a sample. Methods of the present invention include linking universal nucleic acid segments into a single molecule in a linking reaction dependent on a target nucleic acid of interest. A variety of universal segment linking strategies are provided, including preamplification by polymerase chain reaction, ligation-based strategies, reverse transcription and linear polymerase extension. Linking the universal segments into a single molecule generates a tagged target nucleic acid which is detected in a manner dependent on an intramolecular interaction between one universal segment and a second portion of the tagged target nucleic acid. In certain embodiments, the intramolecular interaction includes the formation of a hairpin having a stem between a universal segment at one end of the tagged target nucleic acid and a second universal segment at the opposite end of the tagged target nucleic acid. A variety of detection formats are provided, including solution-phase and surface-based formats. The methods and compositions are well-suited for highly multiplexed nucleic acid detection, and are applicable for the detection of any target nucleic acid of interest in both research and clinical settings.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a non-provisional utility patent applicationclaiming priority to and benefit of the following prior provisionalpatent applications: U.S. Ser. No. 61/302,876, filed Feb. 9, 2010,entitled “Methods for nucleic acids detection using universal hairpinforming primers and its applications” by Eugene Spier; and U.S. Ser. No.61/308,982, filed Feb. 28, 2010, entitled “Methods for nucleic acidsdetection using universal hairpin forming primers” by Eugene Spier. Eachof these provisional applications is incorporated herein by reference inits entirety for all purposes.

BACKGROUND OF THE INVENTION

Traditional methods to detect DNA in a sample using polymerase chainreaction (PCR) detect double-stranded DNA, e.g., SYBR (Higuchi, U.S.Pat. No. 5,994,056) or use probe based detection. Examples of the lattermethod include TAQMAN™ (Gelfand et al., U.S. Pat. Nos. 5,210,015 and5,487,972 and Livak et al., U.S. Pat. No. 5,723,591) and molecularbeacons (Tyagi et al., International Publication No. WO 1995/013399). InTAQMAN™, a probe molecule that includes a fluorescent label and aquencher hybridizes to a PCR amplification product and is digested bythe 5′ exonuclease activity of a polymerase. The 5′ exonuclease activityreleases the fluorescent label from the probe, thereby separating thefluorescent label from the quencher and permitting detection of thefluorescent signal. One application of TAQMAN™ detection that finds usein SNP genotyping is described by Livak et. al (U.S. Pat. No.5,538,848). The molecular beacons method utilizes a probe moleculehaving a stem-loop structure that keeps a fluorescent label and aquencher in close proximity. The probe molecule opens up upon binding toits complementary target, decreasing the quenching so that thefluorophore emits more light proportional to the number of targetmolecules amplified at a given cycle.

Whitcombe et al. describe the “Scorpion” method for DNA detection (U.S.Pat. No. 6,326,145 and 2005/0164219). This method employs a PCR primerwith a 5′ tail that has a dye and a quencher. The 3′ end of the primermatches the target DNA in the sample, but the 5′ tail is complementaryto the target amplicon this primer generates upon extension. Thus, theprimer extension product forms a hairpin during PCR resembling ascorpion, and a fluorescent signal can be detected using the 5′ nuclease(e.g., TAQMAN™), molecular beacon or other method. Whitcombe et al.(Nature Biotechnology (1999) 17:804-807) describe unimolecular scorpiondetection using “sunrise” primers that form a hairpin that brings a dyeand a quencher close to each other; after primer extension, anotherhairpin forms with the dsDNA stem that increases the distance betweenthe dye and the quencher. Thelwell et al. (Nucleic Acid Research (2000)28: 3752-61) compared scorpion “molecular beacon” and 5′ nucleasedetection with TAQMAN™ and showed that unimolecular scorpions givestronger signal than bimolecular TAQMAN™. Solinas et al. (Nucleic AcidsResearch (2001) 29(2):e96) compared molecular beacon and duplex scorpionwith the quencher on a separate oligo and found that the latter providedlarger signal difference between the quenched and unquenched states. Theadvantage of the scorpion method is that there is no separate probemolecule required to anneal to the amplicon (bimolecular interaction)and intramolecular hairpin formation has kinetic and thermodynamic(using the same probe versus hairpin stem sequence) advantages overintermolecular interactions. Using the scorpion method, hairpinformation will nearly always precede PCR primer annealing, whereasprimer annealing often precedes probe to template annealing, leading toa decrease in detectable signal. For a review of various approaches forgenerating a detectable signal during quantitative PCR, as well asCycling Probe Technologies (CPT)-based FEN, invader-type signalgeneration, and 5′ nuclease FRET signal, see Kutyavin, InternationalPublication No. WO 2007/127999.

Methods that utilize universal primers and separate probes for nucleicacid detection have been described. For example, see Whitcombe et al.(U.S. Pat. No. 6,270,967) and Anderson et al. (U.S. Pat. No. 7,601,821).A drawback of these methods is that they are prone to generatenon-specific signal. In these methods, a fluorescent signal is generatedeven if a single primer is involved in amplification. These methodsgenerally use a multiplex pre-amplification (encoding), so inevitablyprimer dimers and non-specific amplifications occur. Even after thedilution that usually follows the pre-amplification step, thesenon-target amplicons and unused pre-amplification primers are stillpresent in the mixture. Single primer specificity makes it moredifficult to develop applications with “clean NTC” (“non templatecontrol”) signal.

The drawbacks of previously described detection methods that utilizetarget-specific primers and probes are cost and logistics. Scorpionapproaches require a specific dual-labeled primer for each targetnucleic acid (e.g., see U.S. Pat. No. 6,326,145), making theseapproaches quite expensive, especially when detection of multipletargets is required. For example, there are approximately 25,000 humangenes and more than 10 million human single nucleotide polymorphisms(SNPs), and researchers often need to measure expression levels ordetermine the genotypes for tens, hundreds, or sometimes even thousandsof targets. Furthermore, each user or research team/project generallyrequires a different set of genes/SNPs.

Several dyes are currently available as amidites, e.g., VIC and FAM. Itis relatively inexpensive to manufacture oligonucleotides (“oligos”)with these dyes, as all of the oligo synthesis steps are performed on acolumn. There is a broad choice of fluorescent dyes that can beincorporated into oligos, but they require off-column dye attachment,e.g., CY3™, CY5™, and TEXASRED™. This makes oligo manufacturing moreexpensive, and often impractical, when each target requires a customsynthesis. Universal detection assays described hereinbelow amortize theoligo synthesis cost over multiple customers and experiments making itcost-effective to use the “off-column” dyes. Several commercial vendorspreload assays in wells to simplify lab work for customers, e.g.,TAQMAN™ low-density arrays and BIOTROVE™ from Life Technologies andSUPERARRAYS™ from Qiagen. Most customers require a custom set of assays,making manufacturing logistics for target-specific assays complicated.As described in detail herein, the present invention permits universalsets of assays for detecting any set of nucleic acid targets, making itmuch easier to offer preloaded assays: the same preloaded assays can beused to detect any set of targets. In addition, the present inventionseparates the encoding and detection steps, providing flexibility todetect multiple targets in the same color (in the same or differentreaction volume), see, e.g., the examples for detecting trisomy 21 andHIV drug resistance mutations hereinbelow. Finally, unlike otheruniversal detection methods, the present invention has two primer plusvirtual probe specificity, rather than only two primer specificity forpreviously described universal detection formats.

Exiqon and Roche have developed the UNIVERSAL PROBELIBRARY™ (UPL) forgene expression that uses a set of 165 short 8-9 base universal probes(see Roche Applied Science website). These short probes have severallocked nucleic acids (LNA, U.S. Pat. No. 6,670,461) that exhibit a highmelting temperature when hybridized to DNA, in spite of being short. Theuniversal probes also have normal DNA bases at the 5′ end so that theycan be cleaved by the 5′-nuclease activity of a polymerase during PCR.The universal probes limit possible PCR primer locations in genes.However, the 165 universal probe set has sufficient occurrences in mostmRNA sequences to enable choosing PCR primers such that at least one ofthe 165 universal sequences can be located between the two primers. Thedetection assays, however, are not universal, as each gene expressionassay requires two target-specific primers.

Due to the above-noted drawbacks of current detection strategies (e.g.,specificity, cost, manufacturing logistics, and the like), there is aneed for more specific, flexible, and cost-effective methods of nucleicacid detection. The present invention meets these and a variety of otherneeds.

SUMMARY OF THE INVENTION

The present invention generally provides methods and compositions fordetecting the presence or amount of one or more target nucleic acids ina sample. Methods of the present invention include linking universalnucleic acids segments into a single molecule in a linking reactiondependent on a target nucleic acid of interest. A variety of universalsegment linking strategies are provided, including preamplification bypolymerase chain reaction, ligation-based strategies, reversetranscription and linear polymerase extension. Linking the universalsegments into a single molecule generates a tagged target nucleic acidwhich is detected in a manner that uses an intramolecular interactionbetween a segment of a first universal primer and a segment of thetagged target nucleic acid. In certain embodiments, the intramolecularinteraction includes the formation of a hairpin having a stem between auniversal segment at one end of the tagged target nucleic acid and asecond universal segment at the opposite end of the tagged targetnucleic acid. A variety of detection formats are provided, includingsolution-phase and surface-based formats. The methods and compositionsare well-suited for highly multiplexed nucleic acid detection, and areapplicable for the detection of any target nucleic acid of interest inboth research and clinical settings.

In a first aspect, the present invention provides a method of detectingthe presence or amount of a first target nucleic acid in a sample, themethod including linking first and second universal DNA segments into afirst molecule in a linking reaction dependent on the first targetnucleic acid, thereby providing a first tagged target nucleic acid. Thefirst tagged target nucleic acid is PCR amplified using first and seconduniversal primers, each universal primer having a 3′ portion thatanneals to one of the two universal DNA segments. The 3′ portion of thefirst universal primer anneals to the first universal segment and a 5′portion of the first universal primer includes a nucleic acid sequencesubstantially identical to a portion of the first universal DNA segment,a portion of the second universal DNA segment, or a portion of the firsttarget nucleic acid. An amplicon generated upon extension of the firstuniversal primer forms an intramolecular hairpin stem between the 5′portion of the first universal primer and a portion of the ampliconcomplementary to the first universal DNA segment, the second universalDNA segment, or the first target nucleic acid. Alternatively, the firstuniversal primer forms a circular structure upon annealing to the firsttagged nucleic acid that brings the 3′ and 5′ ends of the firstuniversal primer within close proximity to each other. Formation of thehairpin stem or circular structure results or causes a change in a firstdetectable signal, the first detectable signal indicating the presenceor quantity of the first target nucleic acid in the sample.

The first target nucleic acid includes a nucleic acid or nucleic acidfeature selected from: a DNA, an RNA, a mammalian nucleic acid, aprimate nucleic acid, a rodent nucleic acid, a viral nucleic acid, abacterial nucleic acid, an archaea nucleic acid, a cDNA, a cDNAcorresponding to a short RNA, a bisulphite treated DNA, a geneticvariant, a mutation or insertion that confers drug resistance, a somaticmutation, a polymorphism, a single nucleotide polymorphism, a rareallele, a portion of the KRAS gene, a nucleic acid that exhibits avariation in copy number, an intron, an exon, an intron-exon boundary, asplice junction, one or more dinucleotides corresponding to one or moremethylated or unmethylated CpG dinucleotides, a nucleic acid comprisingone or more restriction enzyme recognition sequences, a portion of humanchromosome 21, a portion of the human X chromosome, and a portion of thehuman Y chromosome.

The present invention provides a number of approaches for linking thefirst and second universal DNA segments into a first molecule togenerate the first tagged target nucleic acid. In a first embodiment,the linking reaction includes a PCR reaction that includes: a firstprimer having a 3′ portion that anneals to a first portion of the firsttarget nucleic acid and a 5′ portion including the first universal DNAsegment; and a second primer having a 3′ portion that anneals to asecond portion of the first target nucleic acid and a 5′ portionincluding the second universal DNA segment. Amplification of the firsttarget nucleic acid using the first and second primers links the firstand second universal DNA segments into a single molecule.

The linking reaction can include a ligation reaction, whereoligonucleotides complementary to the first target nucleic acid include5′ and 3′ ends extending beyond the first target nucleic acid, and wherethe oligonucleotides serve as a template to ligate the first and seconduniversal DNA segment to the first target nucleic acid. Otherligase-based strategies are provided. For example, the linking reactioncan include a ligation reaction, where the first universal DNA segmentis ligated to the second universal DNA segment on the first targetnucleic acid, thereby linking the first and second universal DNAsegments together into a single molecule.

Linking the first and second universal DNA segments into a firstmolecule can also be accomplished by reverse transcription. In thisaspect, the reverse transcription reaction includes generating a firstcDNA strand by extending a first primer having a 3′ portion that annealsto a first portion of the first target nucleic acid and a 5′ portionthat includes the first universal DNA segment, where the target nucleicacid is an RNA. A second cDNA strand is generated by extending a secondprimer having a 3′ portion that anneals to a portion of the first cDNAand a 5′ portion that includes the second universal DNA segment.Synthesis of the first and second cDNA strands links the first andsecond universal DNA segments into a single molecule.

Linear polymerase extension can be employed in accordance with thepresent invention to link the first and second universal DNA segmentsinto a first molecule. In this aspect, the first universal DNA segmentis linked to the first target nucleic acid by a polymerase that extendson the template complementary to the first universal DNA segment. Aprimer with a 5′ tail that includes the second universal DNA segmentintroduces the second universal DNA segment, thereby linking the firstand second universal segments into a single molecule. Preferably, the 3′end of the first target nucleic acid is defined, e.g., after digestionby a restriction enzyme.

As will be appreciated, the methods and compositions of the presentinvention are well suited for the detection of multiple target nucleicacids simultaneously, either in the same reaction volume or separatereactions. In accordance with the present invention, the universalsegment linking step can be multiplexed such that multiple targetnucleic acids of different nucleic acid sequences are tagged with thesame or different pairs of universal DNA segments. The different pairsof universal primers are optionally labeled with different dyes, thedifferent dyes having different emission wavelengths, and during thedetection step, the different dyes having different emission wavelengthsare detected separately. Optionally, the target nucleic acids ofdifferent nucleic acid sequences are tagged with the same pair ofuniversal DNA segments. When the universal primers are labeled withdifferent dyes with different wavelengths and are detected separately,the first universal primer optionally includes a 5′ portion that issubstantially identical to a first strand of an internal portion of thetagged target nucleic acid, the second universal primer comprises a 5′portion that is substantially identical to the strand of the internalportion of the tagged target nucleic acid, and amplification of bothstrands of the tagged target nucleic acid is measured independentlyusing two or more colors. According to this aspect, the target nucleicacid can include two closely spaced polymorphisms or methylation sites,where the two closely spaced polymorphisms or methylation sites arehaplotyped using four colors.

The linking step can include linking n and n+1 universal DNA segmentsinto one or more additional molecules in a linking reaction specific toone or more additional target nucleic acids, thereby providing one ormore additional tagged target nucleic acids in a single reaction volume,where n is a third or higher number. For example, the linking step canfurther include linking third and fourth universal DNA segments into asecond molecule in a linking reaction specific to a second targetnucleic acid, thereby providing a second tagged target nucleic acid. Fordetection of the second tagged target nucleic acid, the second taggedtarget nucleic acid can be PCR amplified using universal primersspecific to the third and fourth universal DNA segments, where ampliconsgenerated from amplification of the second tagged target nucleic acidform hairpin stems or circles that result in a second detectable signalthat is distinguishable from the first detectable signal.

The linking reaction can occur in a first reaction location, and theuniversal detection step can occur in one or more different reactionlocations. The one or more reaction locations of the detection step caninclude, e.g., a well, a nano-well, a droplet, an array structure, abead surface, or a flat surface. The one or more reaction locations caninclude an array structure or a bead surface, where the first universalprimer is attached to the array structure or bead surface. Subsequent tothe universal segment linking step, and prior to the detection step, areaction mixture including the product of the linking reaction at thefirst location is optionally transferred to the one or more differentlocations, where the reaction mixture is optionally diluted prior to, orduring, transfer. In a related aspect, the first and second universalprimers are delivered to the one or more different reaction locationsprior to, during, or after the linking reaction mixture is transferredto the one or more different reaction locations. Optionally, the one ormore different reaction locations of the detection step are disposedwithin or upon a detection plate or fluidic device, where the first andsecond universal primers are preloaded into the one or more differentreaction locations.

The first universal primer optionally includes a first label proximal toone end of the 5′ portion of the first universal primer. The firstuniversal primer can include a polymerase blocking unit disposed betweenthe 5′ and 3′ portions of the first universal primer. The first labeloptionally includes a fluorescent dye. The first universal primer caninclude a label quencher or FRET dye disposed proximal to the same endor proximal to an end opposite the 5′ portion of the first universalprimer as compared to the first label, where the label quencher or FRETdye is disposed at an effective quenching or FRET distance from thefirst label. The first detectable signal resulting from hairpin stem orcircle formation can include a change of FRET caused by changing theaverage distance between the first label and the label quencher or FRETdye. Optionally, the label quencher or FRET dye is disposed at aposition on the first universal primer selected from: a position betweenthe 5′ and 3′ portions of the first universal primer, and a position 5′of the junction between the 5′ and 3′ portions of the first universalprimer. Some dyes can generate signal when they are cleaved off from anucleic acid to which they were attached, and where a label quencher isnot required to generate 5′ nuclease-based signal, e.g., see U.S.Publication No. 2007/0020664.

Changing the distance between the first label and the label quencher orFRET dye is optionally performed by a mechanism selected from: removalof the label and/or label quencher or FRET dye from the amplicon orprimer by a nuclease reaction; increasing the distance between the firstlabel and the label quencher or FRET dye, where the label quencher orFRET dye is initially disposed at an effective quenching or FRETdistance from the first label via a double-stranded hairpin stem or arandom coil ssDNA structure in the first universal primer, and where thedistance between the label quencher or FRET dye and the first label isincreased by the intramolecular hairpin dsDNA stem that is formed withinthe amplicon generated upon extension of the first universal primer;melting of an oligonucleotide from the first universal primer, where theoligonucleotide includes the label quencher or FRET dye, where the firstuniversal primer comprises the first label, and where the hairpin stemor circle generated upon extension of the first universal primer meltsthe oligonucleotide from the first universal primer, thereby increasingthe distance between the label quencher or FRET dye and the first label;and forming a hairpin stem or circle that disposes the first label at aneffective FRET distance from a quencher or FRET dye attached to anoligonucleotide that is complementary to the amplicon generated uponextension of the first universal primer.

When changing the distance between the first label and the labelquencher or FRET dye is performed by removal of the label and/or labelquencher or FRET dye from the amplicon or primer by a nuclease, thefirst label is optionally disposed between the 3′ and 5′ portions of thefirst universal primer, where the label quencher or FRET dye aredisposed at the 5′ portion of the first universal primer as compared tothe first label, where the label quencher is removed from the ampliconwith the nuclease, and where the labeled amplicon is detected bycapillary electrophoresis or hybridization to a surface-boundoligonucleotide that includes a region complementary to the amplicon.

According to the methods of the present invention, the first detectablesignal can be measured at every PCR cycle (e.g., real-time PCR), thefirst detectable signal can be detected at several points during PCR,and/or the first detectable signal can be detected as an end-pointsubsequent to PCR (e.g., digital PCR or other end-point detectionmethod). When the first detectable signal is detected as an end-pointsubsequent to PCR, the method can further include counting the number ofwells, nano-wells, droplets, array structures, or beads from which thedetectable signal is generated.

When the universal segment linking step is multiplexed such thatmultiple target nucleic acids of different nucleic acid sequences aretagged with the same or different pairs of universal DNA segments, themultiple target nucleic acids are optionally selected from: a DNA, anRNA, a primate nucleic acid, a rodent nucleic acid, a viral nucleicacid, a bacterial nucleic acid, an archaea nucleic acid, a cDNA, a cDNAcorresponding to a short RNA, a genetic variant, a mutation or insertionthat confers drug resistance, a somatic mutation, a polymorphism, asingle nucleotide polymorphism, a rare allele, a portion of the KRASgene, a nucleic acid that exhibits a variation in copy number, anintron, an exon, an intron-exon boundary, a splice junction, one or moredinucleotides corresponding to one or more methylated or unmethylatedCpG dinucleotides in bisulphite treated DNA, a nucleic acid includingone or more restriction enzyme recognition sequences, a portion of humanchromosome 21, a portion of the human X chromosome, and a portion of thehuman Y chromosome, where the presence and/or quantity of the multipletarget nucleic acids is detected and combined into a diagnostic output.The diagnostic output is optionally diagnostic for fetal aneuploidy,fetal sex, and/or fetal copy number variation, where the fetalaneuploidy, fetal sex, and/or fetal copy number variation is detected bydigitally counting nucleic acid targets indicative of fetal aneuploidy,fetal sex, and/or fetal copy number variation from a maternal bloodsample. The nucleic acid targets indicative of fetal aneuploidy, fetalsex, and/or fetal copy number variation can be detected using a firstdetectable label, and control nucleic acid targets are detected using asecond detectable label, where the ratio of digital counts of the labelsis used to detect fetal aneuploidy, fetal sex, and/or fetal copy numbervariation.

Surface-based detection formats are provided by the present invention.For example, the first universal primer can be attached to a surface,where the label is disposed between the 5′ and 3′ portions of the firstuniversal primer, where the label quencher or FRET dye is disposed at a5′ position relative to the label, where the first or second universalprimer is extended by a polymerase where the 5′ universal segment withlabel quencher or FRET dye hybridizes to the amplicon, and whereextension of the universal primer removes the quencher or FRET dye fromthe surface-bound universal segment, thereby resulting or causing achange in the first detectable signal on the surface. According to thisembodiment, the second universal primer can be in solution or attachedto the surface. Methods other than removal of a quencher by exonucleaseactivity can be used to generate signal on the surface, e.g., FRET basedon molecular beacon, “sunrise primers”, duplex between the surface-boundfirst primer and a separate probe that is disrupted upon primerextension. Signal on the surface can also be measured due to changes inelectrochemical properties on the surface due to the first primerextension (“electrochemical detection”), as reviewed in, e.g., Ye andJu, Sensors (2003) 3:128-145. It will be understood that, in accordancewith the present invention, the detection of target nucleic acids can becarried out using electrochemical detection and othernon-fluorescence-based detection methods.

When the universal segment linking (“encoding”) step is multiplexed suchthat multiple target nucleic acids of different nucleic acid sequencesare tagged with the same or different pairs of universal DNA segments,pools of encoding primers are optionally inventoried or made to order,where the encoding primers are capable of amplifying a nucleic acidtarget selected from a gene, a portion of a gene, a single nucleotidepolymorphism, a nucleic acid target that permits detection of a copynumber variation, a methylation target, an miRNA, and a somaticmutation, and where one or more pools of the encoding primers can beprovided as a superset. Optionally, the superset includes a first poolof encoding primers capable of amplifying somatic mutations, singlenucleotide polymorphisms and/or copy number variants, and where thesuperset further includes a second pool of encoding primers capable ofamplifying one or more cDNAs or miRNAs to determine gene expressionlevels. Both pools can be used on the same biological sample, where theresults of both pools can be combined and analyzed together.

The present invention also provides analyte nucleic acid detectionreaction mixtures that include an analyte nucleic acid including anucleic acid subsequence of interest. The analyte nucleic acid furtherincludes first, second and third tag sequences, the second tag sequencebeing located between the first and third tag sequences. The mixturealso includes a first universal primer comprising a first tag complementsubsequence that is complementary to the first tag sequence, asubsequence that includes the second tag sequence, or a subsequencethereof, and a detectable label (e.g., a fluorescent label). The mixturefurther includes a second universal primer including a sequence thatincludes the third tag sequence or a subsequence thereof. Optionally,the mixture further includes a complementary nucleic acid that includessubsequences complementary to the nucleic acid subsequence of interestand the first, second and third tag sequences. The nucleic acidsubsequence of interest optionally includes a nucleic acid or nucleicacid feature selected from: a DNA, an RNA, a primate nucleic acid, arodent nucleic acid, a viral nucleic acid, a bacterial nucleic acid, anarchaea nucleic acid, a cDNA, a cDNA corresponding to a short RNA, agenetic variant, a mutation or insertion that confers drug resistance, asomatic mutation, a polymorphism, a single nucleotide polymorphism, arare allele, a portion of the KRAS gene, a nucleic acid that exhibits avariation in copy number, an intron, an exon, an intron-exon boundary, asplice junction, one or more dinucleotides corresponding to one or moremethylated or unmethylated CpG dinucleotides, a nucleic acid comprisingone or more restriction enzyme recognition sequences, a portion of humanchromosome 21, a portion of the human X chromosome, and a portion of thehuman Y chromosome.

The first universal primer of the reaction mixtures described aboveoptionally includes a label quencher disposed at an effective quenchingdistance from the label. The label quencher can be located between thefirst tag complement subsequence and the subsequence that comprises thesecond tag sequence, where the label is located on an end opposite thesubsequence that includes the second tag sequence, as compared to thelabel quencher.

When the first universal primer includes a label quencher disposed at aneffective quenching distance from the label, the label is optionallylocated between the first tag complement sequence and the subsequencethat includes the second tag sequence, where the label quencher islocated on an end opposite the subsequence that includes the second tagsequence, as compared to the label. The first universal primer caninclude a polymerase blocking unit disposed between the first and seconduniversal segments.

The reaction mixtures of the present invention can further include:

-   a second analyte nucleic acid that includes a second nucleic acid    subsequence of interest, the second analyte nucleic acid further    including fourth, fifth and sixth tag sequences, the fifth tag    sequence being located between the fourth and sixth tag sequences; a    third universal primer that includes a second tag complement    subsequence complementary to the fourth tag sequence, a subsequence    that comprises the fifth tag sequence, or a subsequence thereof, a    label and a label quencher, where the label of the second universal    label primer is different from the label of the first universal    label primer; and, a fourth universal primer including a sequence    that includes the sixth tag sequence or a subsequence thereof.    Optionally, the sixth tag sequence and the third tag sequence are    the same.

The second nucleic acid subsequence of interest optionally includes anucleic acid or nucleic acid feature selected from: a DNA, an RNA, aprimate nucleic acid, a rodent nucleic acid, a viral nucleic acid, abacterial nucleic acid, an archaea nucleic acid, a cDNA, a cDNAcorresponding to a short RNA, a genetic variant, a mutation or insertionthat confers drug resistance, a somatic mutation, a polymorphism, asingle nucleotide polymorphism, a rare allele, a portion of the KRASgene, a nucleic acid that exhibits a variation in copy number, anintron, an exon, an intron-exon boundary, a splice junction, one or moredinucleotides corresponding to one or more methylated or unmethylatedCpG dinucleotides, a nucleic acid including one or more restrictionenzyme recognition sequences, a portion of human chromosome 21, aportion of the human X chromosome, and a portion of the human Ychromosome. Optionally, the first nucleic acid subsequence of interestcomprises a cDNA sequence corresponding to an mRNA expressed from afirst gene, and the second nucleic acid subsequence of interestcomprises a cDNA sequence corresponding to an mRNA expressed from asecond gene.

Methods in addition to those described above are provided by the presentinvention. For example, provided is a method of detecting an analytenucleic acid, the method including providing a reaction mixture thatincludes an analyte nucleic acid that includes a nucleic acidsubsequence of interest, the analyte nucleic acid further includingfirst, second and third tag sequences, the second tag sequence beinglocated between the first and third tag sequences. The reaction mixtureof the method also includes a first universal primer that includes afirst tag complement subsequence that is similar (e.g., complementary)to the first tag sequence, a subsequence that includes the second tagsequence, or a subsequence thereof, a label, and a label quencher. Thereaction mixture of the method also includes a second universal primerincluding a sequence that includes the third tag sequence or asubsequence thereof. The second step of the method includes annealingthe tag complement subsequence of the first universal primer to thefirst tag of the analyte nucleic acid. The third step includesperforming a first primer extension reaction that extends the firstuniversal primer, where after the extension is complete and dsDNA melts,a hairpin stem forms between first and second portions of the product ofthe first primer extension reaction, where the first portion includesthe second tag sequence, and where the second portion is complementaryto the second tag sequence. The fourth step includes annealing thesecond universal primer to the product of the first primer extensionreaction. The fifth step includes performing a second primer extensionreaction that extends the universal specificity primer, where the secondprimer extension reaction releases the label and/or label quencher fromthe product of the first primer extension reaction. The final step ofthe method includes detecting the label, where signal from the labelindicates the presence of the analyte nucleic acid.

Optionally, the nucleic acid subsequence of interest includes a nucleicacid or nucleic acid feature selected from: a DNA, an RNA, a primatenucleic acid, a rodent nucleic acid, a viral nucleic acid, a bacterialnucleic acid, an archaea nucleic acid, a cDNA, a cDNA corresponding to ashort RNA, a genetic variant, a mutation or insertion that confers drugresistance, a somatic mutation, a polymorphism, a single nucleotidepolymorphism, a rare allele, a portion of the KRAS gene, a nucleic acidthat exhibits a variation in copy number, an intron, an exon, anintron-exon boundary, a splice junction, one or more dinucleotidescorresponding to one or more methylated or unmethylated CpGdinucleotides, a nucleic acid comprising one or more restriction enzymerecognition sequences, a portion of human chromosome 21, a portion ofthe human X chromosome, and a portion of the human Y chromosome.

In one aspect, providing the analyte nucleic acid includes performing apreamplification reaction. According to this aspect, performing thepreamplification reaction optionally includes: annealing a firstpreamplification primer to a target nucleic acid, where the firstpreamplification primer comprises a 3′ portion that anneals to a firstportion of the target nucleic acid, and where the first preamplificationprimer further includes a subsequence complementary (e.g., substantiallysimilar) to the first tag sequence; annealing a second preamplificationprimer to a complementary strand of the target nucleic acid, where thesecond preamplification primer includes a 3′ portion that anneals to thetarget nucleic acid, where the second preamplification primer furtherincludes a subsequence that includes the second tag sequence and asubsequence that includes the third tag sequence; and, PCR amplifyingthe target nucleic acid using the first and second preamplificationprimers to generate a plurality of amplicons that include the analytenucleic acid.

Also provided by the present invention is a method to detect or measurethe amount of one or more target nucleic acids in a sample by theirhybridization to a surface-bound first tailed primer that has a dye anda quencher at a 5′ end of the primer, such that when the first tailedprimer is extended, a 5′ nuclease reaction cleaves off the quencher,generating a change in fluorescent signal on the surface.

The methods and compositions summarized above are described in detailhereinbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a schematic illustration of an example universaldetection format, the format including the formation of a unimolecularhairpin having a stem between universal segments at opposite ends of afirst tagged target nucleic acid.

FIG. 2 provides a schematic illustration of an example universaldetection format similar to that shown in FIG. 1, but where thepositions of the detectable label and label quencher are switched, e.g.,so that labeled amplicons can be detected by capillary electrophoresis(CE) or surface hybridization.

FIG. 3 schematically illustrates an example universal detection formatthat utilizes fluorescence (or Forster) resonance energy transfer.

FIG. 4 provides a schematic illustration of an example universaldetection format, the format including the formation of a unimolecularhairpin having a stem between complementary subsequences present in asingle universal segment at one end of a first tagged target nucleicacid.

FIG. 5 schematically illustrates an example universal detection format,the format including the formation of a unimolecular hairpin having astem between a portion of a first universal segment and a sequencecorresponding to the target nucleic acid of interest.

FIG. 6 schematically illustrates an example universal circle detectionformat.

FIG. 7 provides a schematic illustration of an example surface-baseduniversal detection format that utilizes emulsion PCR.

FIG. 8 schematically illustrates an exemplary surface-based universaldetection format that utilizes bridge PCR.

FIG. 9 provides a schematic illustration of a second surface-baseduniversal detection format that utilizes bridge PCR.

FIG. 10 schematically illustrates a universal segment linking strategythat includes preamplification of a target nucleic acid with primershaving 3′ ends specific to the target nucleic acid and 5′ ends havinguniversal segments.

FIG. 11 schematically illustrates a ligation-based universal segmentlinking strategy.

FIG. 12 provides a schematic illustration of a second ligation-baseduniversal segment linking strategy.

FIG. 13 schematically illustrates a strategy for linking universalsegments into a single molecule based upon reverse transcription.

FIG. 14 provides a schematic illustration of a universal segment linkingstrategy based upon linear extension.

FIG. 15 schematically illustrates an example universal detectionapplication for detecting nucleotide polymorphisms.

FIG. 16 provides a schematic illustration of an example universaldetection application for detecting gene expression.

FIG. 17 schematically illustrates an example universal detectionapplication for simultaneously determining copy number variation andgene expression.

FIG. 18 schematically illustrates an example universal two-stranddetection application.

FIG. 19 provides a schematic illustration of an example encoding stepfor universal detection of methylation status of two CpG dinucleotidesin bisulphite treated DNA.

FIG. 20 schematically illustrates an example encoding step for universaldetection of the presence or amount of one or more short RNAs in asample.

FIG. 21 schematically illustrates an example encoding step and universaldetection of mutations that confer resistance to one or more HIVprotease inhibitors.

FIG. 22 provides a schematic illustration of an example multiplexencoding and detection in accordance with the present invention.

FIG. 23 schematically illustrates an example strategy for detectingtrisomy 21 via digital PCR in accordance with the present inventionusing universal middle tags.

FIG. 24 provides a schematic illustration of a second strategy fordetecting trisomy 21 via digital PCR in accordance with the presentinvention using naturally occurring short middle tag sequences.

FIG. 25 schematically illustrates an example strategy for detecting avariety of target nucleic acids, the results from which can be combinedinto a diagnostic output or treatment outcome.

FIG. 26 schematically illustrates an example nucleic acid detectionreaction mixture in accordance with the present invention.

DETAILED DESCRIPTION

Overview

The present invention is broadly applicable to any application in whichone desires to detect or measure the amount of one or more targetnucleic acids in a sample of interest. Generally, the present inventionis directed to methods and compositions for detecting and/or measuringthe amount of a target nucleic acid in a sample using a set of universalPCR primers with sequences that do not hybridize to the target nucleicacid of interest. In a preferred aspect, the invention provides a methodfor detecting a target nucleic acid that includes tagging the targetnucleic by linking first and second universal segments at the two endsof a molecule in a way that depends on the target nucleic acid, and PCRamplifying the tagged target nucleic acid using universal primers thathybridize to the universal segments. In this aspect, regions of the PCRamplicons corresponding to the first and second universal segments formintramolecular hairpin stems, which hairpin stem formation is aprerequisite for detection of the nucleic acid, e.g., by removal of alabel, label quencher and/or FRET dye from the amplicon. This and otheraspects of the invention are described in detail hereinbelow.

This preferred aspect of the invention constitutes a significantimprovement over previous nucleic acid detection methods. First, themethod provides exceptionally clean results (e.g., clean non-templatecontrols) due to its two-primer and virtual probe (middle universal tag)specificity. Signal is detected only when the three universal segmentsare present in the tagged target nucleic acid. This is in contrast toprior methods in which signal is generated when only two primers areinvolved in amplification, e.g., SYBR-green. Second, the detectionmethods described herein are more cost effective and practical thanprevious approaches. The universal detection primers need only be madeonce or can be made in very large batches, because the primers arecapable of detecting different target nucleic acids of interest,amortizing their cost over tens, hundreds, thousands or even millions ofsamples. This feature also makes it practical to achieve highermultiplex detection using more detectable labels/dyes, e.g., one cangenotype two [three] SNPs (four [six] alleles) using four [six]distinguishable fluorescent colors in a well. Third, the universalprimers can be preloaded on a plate (e.g., an integrated fluidic chip),making the method more convenient to use. In accordance with theuniversal primer detection system of the present invention, pre-loadeduniversal assays can be provided for detecting any set of targets, e.g.,SNPs, genes, chromosomes, cDNAs, copy number variants, miRNAs,methylated/non-methylated regions, and the like. Fourth, the methodpermits greater variety in terms of “number of assays x number ofsamples” options for fixed format plates. These and other aspects andadvantages of the present invention are set forth in greater detailbelow.

The present invention provides methods and compositions for detection ofnucleic acids that utilizes artificial universal tagging sequencesrather than traditional direct detection of DNA/RNA using primers andprobes matching the target nucleic acids. Target nucleic acids are onlyused to specifically connect distinct universal tagging sequences into asingle molecule during the first encoding step. The detection is basedon the intra-molecular Watson-Crick base-pairing (a hairpin stem)between the two universal tags in the amplicon. The base-pairing betweenthe two universal tagging sequences enables detection of nucleic acidsthat preserves “three tag” detection specificity: spuriousamplifications and primer dimers have a low probability of forminghairpins. Also provided is universal surface solid-phase detection forencoded nucleic acid targets.

Exemplary Universal Detection Formats

A variety of universal detection formats are provided by the presentinvention. A first example format is schematically illustrated inFIG. 1. As shown, step (a) includes priming on both strands of a firsttagged nucleic acid using first universal primer 100 and seconduniversal primer 102. A 3′ portion (Ai) of the first universal primeranneals to first universal segment 104, while a 3′ portion (Ci) of thesecond universal primer anneals to portion C′i of second universalsegment 106. The first universal primer includes detectable label D1 ator near the 5′ end of the first universal primer, 5′ portion (Bi) and alabel quencher or FRET dye (Q). Optionally positioned proximal to thelabel quencher or FRET dye is a polymerase blocking unit, e.g., HEG(hexethylene glycol), THF (tetrahydrofuran), or any other blocker knownin the field in case the presence of the quencher or FRET dye is notsufficient to stop the polymerase extension. For example, primers with aHEG or Sp-18 polymerase blocking unit are commercially available, e.g.,from BioSearch Technologies. For the sake of simplifying the figuresherein, polymerase blocking units are not shown. However, it will beunderstood that a polymerase blocking unit is optionally positioned inclose proximity to quencher “Q” in the middle portion of the universalprimers. A polymerase blocker may not be required if the 5′-tail thatfolds into a stem has one or more bases at the 5′ end that are notcomplementary to the middle universal tag sequence, so that the hairpinformed by the opposite strand of DNA (with the 3′-end at the end of thestem) is not extendable during PCR. One can also design a small hairpininto the 5′ portion of the primer 100, so that the dye and the quencherare brought closer together, similar to “Sunrise” primers and probes toimprove quenching and decrease background fluorescence. For example, seeU.S. Pat. Nos. 5,866,336 and 6,270,967.

PCR amplification results in double stranded product 108. In thisexample, a polymerase blocking unit positioned proximal to the labelquencher or FRET dye prevents a polymerase from copying the 5′ portion(Bi) of the first universal primer, such that the bottom strand ofproduct 108 cannot form a hairpin when it becomes single-stranded.Formation of such a hairpin would result in the 3′ end of the stemannealing to the amplicon such that polymerase extension of this 3′ endwould terminate the PCR reaction.

Hairpin formation is shown at step (c) of FIG. 1. Product 108 is melted(e.g., by raising the temperature to approximately 95° C.) to separatethe upper strand from the lower strand, and when the temperature issubsequently decreased, the upper strand of product 108 forms a hairpinhaving a stem between 5′ portion (Bi) of the first universal primer andportion B′i at the opposite end of the strand. Also at step (c), thesecond universal primer anneals to a complementary portion of the upperstrand. Intra-molecular hairpin formation occurs rapidly and is drivenby thermodynamics: the free energy is determined by stem length,GC-content and loop length. It is important that the melting temperature(Tm) of the hairpin be significantly higher (e.g., approximately 10° C.or higher) than the Tm of the second universal primer 102. This way,when the temperature is decreased, nearly 100% of the molecules willform the hairpin before the second universal primer anneals and isextended. Upon extension of the second universal primer at step (d), 5′nuclease activity of the polymerase cleaves the detectable label D1 fromthe 5′ end of the amplicon, thereby increasing the distance between thelabel and the quencher or FRET dye and permitting detection of thelabel. A wide variety fluorescent dyes are known in the art andcommercially available, e.g., FAM, TET, JOE, VIC, HEX, CY3, TAMRA,TexasRed, CY5, ROX and many other dyes and quenchers can be used, e.g.,MGB-NFQ, BHQ-[0123], ZEN quencher from IDT.

The present invention provides approaches for signal generationsubsequent to hairpin formation in addition to 5′ nuclease activity.Alternative methods previously described include “molecularbeacons”-type detection, which does not require 5′ exonuclease activity.Rather, a fluorescent signal is generated when the hairpin forms,increasing the spatial distance between the dye and the quencher,thereby decreasing quenching and increasing the fluorescent signal. Fora comparison between scorpion “molecular beacon”, scorpion 5′ nucleaseand bimolecular 5′ nuclease (TAQMAN™ probe) detection methods, seeThelwell et al., Nucleic Acids Research (2000) 28(19): 3752-61.

Because the 5′ Bi tails of the detection primers in FIG. 1 are notcopied during PCR, they can contain any modified bases that are known inthe art. These may include, PNA and LNA, which make dsDNA more stableand thus enable shorter hairpin stems. If these are used at the very 5′end of the Bi primers, they will make the probes resistant to the 5′nuclease cleavage, thus permitting the use of polymerases with theexonuclease activity and generating the molecular beacons-type (“probestretching”) signal described above. Similarly, 2-amino-adenosine isknown to increase the stability of the T-A pairs, again permittingshortening of the stems. RNA bases in the Bi parts will lead to theRNA-DNA hairpin stems that are more stable than DNA-DNA. ZEN quencherfrom IDT also stabilizes dsDNA enabling shortening of stems. Inaddition, uridines, THF, oxo-G and other enzymatically cleavable bases(e.g., UDG, EndoV, Fpg, etc.) can be used to measure the 100% primerdigestion signal after the detection PCR. This signal can be comparedwith the end-point detection signal and serve as a positive control.

Modified bases that can be copied by polymerases can be used in the Aiparts and in the Ci primers (referring to FIG. 1), e.g.,2-amino-adenosine can stabilize priming. These modified bases usuallyhave a weaker affinity to the polymerases and thus can decrease thelikelihood of primer dimer formation (e.g., as described in U.S. Pat.No. 6,794,142). The modified bases increase the cost of the universalprimers, but because primers are amortized over hundreds of thousands oreven millions of samples, the increase per sample is negligible. Themodified bases described above are well known in the art, e.g., GlenResearch catalog website or TriLink Biotechnologies product websiteoffers a broad range of modified bases.

The present invention also provides a detection scheme similar to thatshown in FIG. 1, but where the positions of the detectable label andquencher or FRET dye on the first universal primer are switched. Thisembodiment, which is particularly useful for applications whereretention of the detectable label on the amplicon is desirable, isschematically illustrated in FIG. 2. As shown, step (a) includes primingon both strands of a first tagged nucleic acid using first universalprimer 200 and second universal primer 202. A 3′ portion (Ai) of thefirst universal primer anneals to first universal segment 204, while a3′ portion (Ci) of the second universal primer anneals to portion C′i ofsecond universal segment 206. The first universal primer includes alabel quencher or FRET dye (Q) at or near the 5′ end of the firstuniversal primer, 5′ portion (Bi), and detectable label D1. PCRamplification results in double stranded product 208. Universal hairpinformation is shown at step (c). Upper strand of product 208 forms ahairpin having a stem between 5′ portion (Bi) of the first universalprimer and portion B′i at the opposite end of the strand. Upon extensionof the second universal primer at step (d), 5′ nuclease activity of thepolymerase cleaves the quencher or FRET dye from the 5′ end of theamplicon, thereby increasing the distance between the quencher or FRETdye and the label and permitting detection of the label. In thisexample, a polymerase blocking unit is positioned proximal to label D1,so that the label is not cleaved from the amplicon.

When practicing the “switched” format in which the detectable label is afluorescent dye disposed at an internal portion of the first universalprimer, it is preferable that the number of guanine (“G”) bases in theimmediate vicinity of the detectable label be minimized or avoidedaltogether. This is because guanine is capable of quenching fluorescenceemitted from certain fluorophores, potentially reducing the strength ofthe detected signal from the label.

Retaining the detectable label on the amplicon can be useful in a numberof applications, e.g., when it is desirable to detect the amplicon viacapillary electrophoresis (CE), or on a surface or bead (e.g., in thecase of surface- or bead-based DNA arrays). Step (e) of FIG. 2schematically illustrates capillary electrophoresis (CE) detecting peaksat certain amplicon lengths. Step (f) schematically illustrates atarget-specific DNA array, where the labeled amplicon generated at step(d) hybridizes to complementary nucleic acids attached to the arraysurface, permitting detection of the amplicon as color or signal on thearray. The complementary nucleic acids attached to the array surface canbe universal as shown in FIG. 2( f) or target-specific.

Both CE and DNA arrays allow high multiplex endpoint detection oflabeled amplicons. For example, amplicons generated using the “switched”format with size ranges between, e.g., 70 and 200 bases, can be detectedas fluorescent CE peaks in four or more dyes. Assuming that robust peakseparation requires approximately 10 bases between peaks in the samecolor, it is possible to detect, e.g., 4 dyes×(200−70)/10=52 peakscorresponding to 26 biallelic SNPs, or detecting the presence or absenceof 52 DNA targets. DNA arrays have essentially unlimited detectionmultiplicity.

The “switched” format can be used for a combinedscreening/identification in cases where the majority of samples areexpected to be negative. For example, in a diagnostic or epidemiologicalapplication, one can detect the presence of any of 52 differentpathogens or 52 alleles using real-time (or end-point in a well) PCRdetection. The majority of samples are expected to be negative, but ifone of the dyes shows signal in a qPCR well for a given sample, theproducts of this amplification can be loaded onto a CE lane or DNAarray. The exact nature of the pathogen or alleles can be determinedbased upon the size of the DNA fragment on CE, or known DNA sequencespotted at a specific location on the array.

A third universal detection format, which utilizes fluorescence energyresonance transfer (FRET) to generate a detectable signal (or a changein detectable signal), is schematically illustrated in FIG. 3. FRET canbe useful to expand the number of detectable fluorescent colors withlonger (red) wavelengths. As shown, step (a) includes priming on bothstrands of a first tagged nucleic acid using first universal primer 300and second universal primer 302. A 3′ portion (Ai) of the firstuniversal primer anneals to first universal segment 304, while a 3′portion (Ci) of the second universal primer anneals to portion C′i ofsecond universal segment 306. The first universal primer includes a dye(D1) disposed at or near the 5′ end of the primer, but does not includea quencher. D1 does not generate signal, because the light in theinstrument does not excite the D1 dye. In addition, there is a separateprobe molecule with a dye (D2) that is excited by the light in theinstrument. PCR amplification results in double stranded product 308.

During the detection reaction shown at step (c) of FIG. 3, a hairpinforms and the probe molecule having dye D2 anneals to portion E′i of theamplicon, such that dyes D1 from the stem and D2 from the probe moleculecome into close proximity to each other. D2 emits light shifted to thered (longer wavelength) and excites D1, resulting in the generation of adetectable FRET signal. It is important that the probe molecule have ahigher melting temperature than the second universal primer, in order toprovide sufficient time to detect/measure the FRET signal before thesecond universal primer extends.

The detection reaction shown at step (d) of FIG. 3 is a variation ofthat shown at step (c). In particular, the first universal primer canhave dye D1 disposed between subsegments Ai and Bi, and the probemolecule having dye D2 anneals to portion B′i of the amplicon. Annealingof the probe molecule brings dyes D1 and D2 into close proximity to eachother, resulting in the generation of a detectable FRET signal.

In one aspect, polymerases without exonuclease activity, but withstrand-displacing activity, are used for the FRET detection.Alternatively, polymerases with 5′ exonuclease activity andstrand-displacement activity can be used, but preferably when the probeis resistant to nuclease digestion. For example, PNA, LNA or othermodifications can be used to confer resistance by the probe molecules tonuclease digestion. The present invention also provides FRET and 5′nuclease detection in the same well. According to this embodiment, asthe temperature is decreased from the denaturation temperature (e.g.,95° C.) during PCR, the FRET signals due to their thermodynamic/temporalnature are measured first, and the signal generated from 5′ nuclease ismeasured second.

FIG. 4 schematically illustrates a fourth example detection format inwhich a hairpin step forms between complementary regions on the sameside of the first tagged target nucleic acid. The tagged target nucleicacid is amplified using first universal primer 400 (includingD1-B′i-Q-Ci-3′) and second universal primer 402 (including Ai). D1 is adetectable label and Q is a quencher and polymerase blocker. A 3′portion (Ci) of the first universal primer anneals to first universalsegment 404, while a 3′ portion (Ai) of the second universal primeranneals to second universal segment 406. PCR amplification results indouble stranded product 408. After product 408 is melted and thetemperature is decreased, a hairpin forms with the stem between Bi andB′i. A signal is generated by the 5′ nuclease activity of a polymeraseas it extends primer Ai.

The present invention also provides detection formats where asubsequence of one of the universal detection primers forms a hairpinstem with a sequence corresponding to the target nucleic acid. Thisformat is schematically illustrated in FIG. 5. As shown, the taggedtarget nucleic acid is amplified using first universal primer 500(including D1-Bj-Q-Ai-3′) and second universal primer 502 (includingCi). D1 is a detectable label and Q is a quencher optionally associatedwith a polymerase blocker. A 3′ portion (Ai) of the first universalprimer anneals to first universal segment 504, while a 3′ portion (Ci)of the second universal primer anneals to second universal segment 506.PCR amplification results in double stranded product 508. After product508 is melted and the temperature is decreased, a hairpin forms with thestem between Bj and B′j, where B′j is a sequence corresponding to thetarget nucleic acid. In this example, a signal is generated by removalof the label from the amplicon by the 5′ nuclease activity of apolymerase as it extends primer Ci.

A further example detection format involving the formation of auniversal circle is schematically illustrated in FIG. 6. The taggedtarget nucleic acid is amplified using universal primer 600 (includingD1-Bi-Q-spacer-x-Ci-3′) and second universal primer 602 (including Ai),where D1 is a detectable label, Q is a quencher and “x” is a polymeraseblocking unit. A 3′ portion (Ci) of the first universal primer annealsto first universal segment 604, while a 3′ portion (Ai) of the seconduniversal primer anneals to second universal segment 606. In thisexample, extension of the first universal primer at step (b) generates asignal by 5′ exonuclease cleavage of D1 and separation of D1 from thequencher.

As will be appreciated, the universal detection methods provided by thepresent invention include surface-based detection formats. Surfaces uponwhich detection can occur include the surface of a well (e.g., anano-well), an array structure (e.g., a reaction region on an array), abead surface, and the like. One example of surface-based detectionprovided by the present invention is universal detection by emulsion PCR(ePCR). Emulsion PCR is known in the art and involves the isolation ofDNA molecules along with primer-coated beads in aqueous droplets withinan oil phase.

An example of universal detection by emulsion PCR is schematicallyillustrated in FIG. 7. As shown, first universal primer 700 and seconduniversal primer 702 are attached to bead 704. The first universalprimer includes Q-B1-D1-spacer-[surface]-spacer-A1-3′ and the seconduniversal primer includes Q-B2-D2-spacer-[surface]-spacer-A2-3′, whereD1 and D2 are detectable labels (e.g., fluorescent dyes) and Q is alabel quencher. The ePCR micro-reactor (droplet 706) has a bead and zeroor one or more of each tagged target nucleic acid molecules for eachcolor, e.g., tagged target nucleic acid A1-B1-C is shown inside thedroplet, while A2-B2-C is outside the droplet. Third universal primer708 (including C) and PCR reagents are inside the droplet. As shown at(c), during ePCR, the A1-bead primer extends and portion B1 of the firstuniversal primer anneals to B′1 of the extended primer. As shown at (d),when the third universal primer (C) extends, quencher Q is cleaved off,D1 becomes unquenched, and D2 remains quenched. D1 and/or D2 signalindicates if one (or both) tagged target nucleic acids were present inthe droplet.

Universal primers of the present invention can be attached, e.g., to abead surface or a flat surface. The dyes are quenched, but when thesurface-bound primers are involved in PCR, the 5′ nuclease reactioncleaves off the quencher and generates detectable signal. The primerscan be attached to the beads using standard methods known in the art,e.g., primary amino groups binding to slides with activated carboxylgroups. For example internal Amino Modifier C6-dT or UNI-LINK™ modifiedbases can be used to attach the first universal primers to the surface.The total number of attached primers depends on the bead size. Smallerbeads, e.g., approximately 1 micron beads can accommodate hundreds ofthousands of primers. Long spacers, e.g., several Ts or any other randomDNA sequences, or so called “spacers”, e.g., spacer 9, spacer 12Integrated DNA Technologies catalog website, “Modifications”) can beused so that both 3′ and 5′ ends of primers are far enough from thesurface to participate in the PCR extension and 5′ nuclease reactions,respectively (e.g., see FIG. 7( a)). An ePCR reaction is prepared bymixing the appropriate number of beads and volumes of oil, surfactants,etc. and PCR master mix that includes the second universal primer (e.g.,primer 708 in FIG. 7) and the tagged DNA with universal ends (e.g., FIG.7( b)). Further details regarding the attachment of various reactioncomponents, e.g., primers, probes, templates, and the like, aredescribed, e.g. in Mitterer G., Schmidt W. M. (2006) Methods MoI. Biol,345: 37-51; Fedurco M. et al (2006) Nucleic Acids Res., 34: e22; KojimaT. et al (2005) Nucleic Acids Res., 33: e150; Mercier J. F., Slater G.W. (2005) Biophys. J., 89: 32-42; Mercier J. F. et al (2003) Biophys.J., 85: 2075-2086.

For digital PCR, each ePCR micro-reactor (e.g., droplet, FIG. 7( b))with a bead has on average less than one copy of the pre-amplifiedtarget molecule for each detection color, so that only a subset of beadswill generate signal in each color and each bead can have signals inmore than one color. During ePCR, a single pre-amplification moleculeexponentially amplifies, e.g., A1-B1-C at FIG. 7( b), drivingapproximately 10⁵ or more A1-bead primer extensions (e.g., see FIG. 7(c)), and the 5′ nuclease reaction cleaves off the 5′ quencher (see FIG.14( d)). Because the A2-B2-C pre-amplification target was outside of thedroplet (FIG. 14( b)), the bead will have no detectable D2 signal.

After ePCR, the emulsion is broken, the beads are deposited on the slidesurface (or, e.g., 454 PicoTiter Plates), and beads in each color or nosignal are counted. For example, two colors are shown in FIG. 7, butfour colors can be routinely detected in existing instruments, e.g.,SOLID™/454. This can be done, e.g., using a microscope with a CCD. Astraightforward statistical analysis of the ratios of bead counts withcolor and no signal, and between the different colors, will yield veryaccurate relative counts for the number of the tagged molecules. Eachdetection color can correspond to one or several pre-amplificationtargets that are counted together. For example, in the case of trisomy21 detection (e.g., see FIGS. 23 and 24) several chromosome 21 targetsare detected using D1/color 1, autosomal non-chromosome 21 targets useD2/color 2, X-chromosome targets use D3/color 3, and so on.

Similarly, digital counts can be obtained using bridge clusteramplification on the surface with densely attached universal detectionprimers, e.g., see FIGS. 8 and 9 and the accompanying descriptionbelow). For example, the cBot Cluster Generation System from Illuminacan be used to generate isothermal bridge amplification clusters.Alternatively, bridge PCR cycling can be used. Similar to the beadsapproach described above, each cluster is clonal and originates from asingle ssDNA pre-amplified molecule that randomly binds to the A1-3′ orA2-3′ on the slide surface (FIG. 7( a)) for the first extension cycle.Bridge amplification gives two options in terms of color detection: aplain “C” universal primer is “densely” attached to the surface so thatit is involved in “bridge amplification” with any of the labeleduniversal primers, generating cluster signal in the color thatcorresponds to the specific pre-amplification target. Alternatively,bridge amplification can use two labeled primers based on the two-stranduniversal detection (e.g., see FIG. 18 below), so that each clustergives a two-color signal. The latter approach can detect more targets ascompared to single color detection. For example, four universal primerslabeled in four colors can detect six different targets: 4*3/2=6, allpossible 2-color combination of 4 colors. Five-color detection will havethe ability to detect 5*4/2=10 tagged products.

Commercially available sequencing instruments, e.g., from Illumina(e.g., HiSeq 1000/2000, GAIIx or MiSeq instruments) can be used to readthe detectable signal on slides. The number of clusters in each color(or a pair of colors) is counted, and the numbers indicate how manypre-amplified target molecules were present in the sample.

It should be noted that bridge amplification or bridge PCR can beperformed on beads in addition to flat surface. In this embodiment, theamplification is similar to ePCR, except instead of the universal primer“C” in solution (as in FIG. 7( b)), the “C” primer is attached to thebeads. This “bridge on the bead” approach has the benefit of asignificantly stronger signal, e.g., SOLID™ 1 micron beads haveapproximately 100,000 extended molecules versus approximately 1,000 in abridge amplification cluster. This is because a single tagged DNAmolecule can start multiple clonal clusters on the bead surface in anePCR droplet. This method also allows several types of beads withdifferent labeled universal primers to be implemented. At the same time,the “bridge on the bead” method has features from the bridgeamplification, e.g., it permits two-strand (two color) universaldetection (e.g., as generally shown in FIG. 18).

Another surface-based universal detection format provided by the presentinvention is schematically illustrated in FIG. 8. In this example,universal detection is accomplished via “bridge PCR” or bridgeamplification, where the universal primers are attached to the surfaceof a bead. As shown, first universal primer 800 and second universalprimer 802 are attached to the surface of bead 804. The first universalprimer includes Q-Bi-D1-spacer-[surface]-spacer-Ci-3′ and the seconduniversal primer includes Ai, where Q is a label quencher and D1 is adetectable label, e.g., a fluorescent dye. First tagged target nucleicacid (including Ai-B′i-C′i) anneals to both the Bi and Ci portions ofthe first universal primer, bringing the 3′-end of Ci close to the5′-quencher of Bi. The extension of Ci at step (c) cleaves the quencherand generates fluorescent signal D1. At step (d), the extended ampliconforms a bridge by looping back onto Ai on the surface, and Ai extends toform a complementary strand, thus continuing amplification on thesurface. The first universal primer on the surface is shown attached inthe middle. An alternative configuration in accordance with the presentinvention would be to have a spacer-Ci-3′ primer attached to the bead atthe 5′ end and Q-Bi-D1-spacer-5′ attached at the 5′ end. The ends of thetwo attached primers would come into proximity with each other due totheir attachment to the same bead surface. In this example, the twoparts of the first universal primer are formally two separate oligos,but being bound to the same bead surface they are effectively linkedinto a single molecule where the surface provides a link.

Universal detection via bridge PCR can also be accomplished asschematically illustrated in FIG. 9. As shown, first universal primer900 and second universal primer 902 are attached to bead 904. The firstuniversal primer includes Q-B′i-D1-spacer-[surface]-spacer-Ci-3′ and thesecond universal primer includes spacer-Ai-3′. First tagged targetnucleic acid 906 (Ai-B′i-C′i) anneals to the Ci portion of the firstuniversal primer. At step (b), extension of Ci attaches the complementof the first tagged target nucleic acid to the surface. The extensionproduct (amplicon Ci-Bi-A′i-3′) anneals to both B′i and the Ai portionof the second universal primer on the surface. The extension of Ai atstep (c) causes 5′ nuclease cleavage of the quencher, permittingdetection of a fluorescent signal emanating from dye D1. The spacersattached to the surface are preferably long and flexible/rotatable.

Multiple beads with different intrinsic bead properties can be used formultiplex detection in FIGS. 8 and 9. For example, bead color (Luminexmicrospheres), holographic images (Illumina VERACODE™) or barcodes(Applied BIOCODE™ beads, Affymetrix liquid arrays) can be pooledtogether for the detection step. One can decode both the universal tags“i” on each bead that encode each target based on the intrinsic beadproperties and the surface signal generated by the label on the firstuniversal primer that measures the amount of the tagged target nucleicacid in the sample.

Surface detection for multiplex targets requires attaching universalprimers to beads or surfaces (FIGS. 8 a, 9 a). Bead encoding can beperformed by the bead vendor: each type of bead with different colors orbarcodes is combined with specific tags Ai, Bi, Ci. All encoded beadsare pooled together for multiplex detection and the universal pool canbe used to detect or measure any nucleic acid target. Users can mixtheir samples containing target nucleic acids, encoding primers, mastermix and encoded beads and PCR cycle the mixture. First, encoded targetmolecules will be generated and then these molecules anneal to thesurface bound universal primers and generate a signal on the surface(see, e.g., FIGS. 8 and 9). In the alternative two-step detectionmethod, targets are first tagged by linking universal segments together.The tagged/encoded sample is optionally diluted and added to the pooleddecoding beads for detection. After hybridization to the beads, bridgeamplification or PCR generates signal on the bead surface. For thereadout, the beads can be laid on the surface (e.g., VERACODE™),streamed past a detector (e.g., Luminex microspheres) or directly imagedin a well (e.g., Applied BIOCODE™). The barcode on a bead or color of amicrosphere determines the target and the fluorescent signal on thesurface measures the amount of this nucleic acid target in the sample.Thus, as provided by the present invention, multiplex surface detectioncan measure the amounts of multiple targets in a well using a set ofuniversal beads or microspheres.

It will be appreciated that the universal detection formats describedabove are exemplary approaches. Additional universal detection formatsare possible and within the scope of the present invention. It will alsobe appreciated that any of the exemplary universal detection formatsdescribed above can be combined with any of the universal segmentlinking strategies and universal detection applications describedherein. Further, it will be understood that the universal detectionformats of the present invention can occur in multiplex, where 2 ormore, e.g., 4 or more, 6 or more, 8 or more, 10 or more, hundreds ormore, or even thousands or more different target nucleic acids ofinterest can be detected simultaneously in wells (e.g., nano-wells), onbeads, on an array, using integrated fluidics chips (IFC), and the like.

Exemplary Universal Segment Linking Strategies

The above section entitled “Exemplary Universal Detection Formats”describes universal detection approaches that utilize a first taggedtarget nucleic acid of interest. The present section describes exemplarystrategies for linking first and second universal DNA segments into asingle molecule in a linking reaction dependent on a first targetnucleic acid of interest, thereby providing the first tagged targetnucleic acid. It will be appreciated that multiple different targetnucleic acids can be “encoded” with different pairs of universalsegments, such that the target nucleic acids can be differentiallydetected (e.g., using any of the universal detection formats describedabove) based upon the particular pair of universal segments associatedwith each different target nucleic acid.

A first exemplary universal segment linking strategy involves“preamplification” of the target nucleic acid of interest with PCRprimers that include a 3′ portion specific to the target nucleic acidand a 5′ universal segment. Linking first and second universal segmentsby preamplification is schematically illustrated in FIG. 10.Target-specific 3′ ends of primers are shown as black arrows. “Primes”indicate complements: e.g., B′1, is complementary to B1. The 3′ ends ofamplicons can have a non-template 3′ “+A-addition”. As shown, firstprimer 1000 has a 3′ portion that anneals to a first portion of firsttarget nucleic acid 1002 and a 5′ portion having a first universalsegment (A1). Second primer 1004 has a 3′ portion that anneals to asecond portion of first target nucleic acid 1002 and a 5′ portion havinga second universal segment (B1-C1). PCR amplification yields amplicon1006, which can be detected using any of the example universal detectionformats described above, e.g., the universal detection format providedin FIG. 1. In FIG. 1, for example, each detection well has at least twoprimers. The 3′ ends of these primers are complementary to all or aportion of the 5′ ends of the pre-amplification primers. Referring toFIG. 1, at least one primer has a 5′ tail with a dye and a quencher thatis identical to the middle universal part of the oppositepre-amplification primer (B_(i) in FIG. 1).

Preamplification can consist of a multiplex PCR that uses a lowconcentration (e.g., 10-100 nM) of primers with target-specific 3′ endsand universal 5′ tails that “encode” each target to be detected. Thenumber of targets in the multiplex can depend on the number offluorescent colors to be detected in the read-out device during theuniversal detection step, e.g., if the read-out platform uses 96 wellsand detects in four colors, a total of N=96*4=384 targets can bemultiplexed in a pre-amplification well. To assure accurate doublingduring pre-amplification PCR, a small number of pre-amplification cycles(C), e.g., C=2-20, can be run. A more typical range of cycles is 10-16.In a preferred aspect, relatively low pre-amplification primerconcentrations are used, e.g., 10-100 nM (typically 30-60 nM) tominimize primer dimer formation. Longer anneal-extend times—typically2-10 minutes—and high polymerase concentrations can be used tocompensate for low primer concentrations.

Pre-amplification primers have 5′ universal segments, represented inFIG. 10 as A1 and B1-C1 for target 1, and A_(N), B_(N) and C_(N) for thelast of N targets. At the end of pre-amplification all N targets havebeen amplified ˜2^C times, e.g., 2^10=1,024 times and universal DNAsegments are linked into a single molecule.

Though it is possible to run pre-amplification and universal detectionPCR in a single closed-tube reaction, a more typical application is torun a separate multiplex pre-amplification followed by optional dilutionand splitting the reaction into multiple detection wells. Performing twoPCR amplifications in separate tubes (or wells, etc.) can be morecumbersome than traditional single-tube real-time PCR. In reality,however, pre-amplification has to be performed anyway when (a) theamount of input DNA/RNA is low, e.g., single or a few cells and/or (b)detection volume is small. Several commercial platforms, e.g., BIOTROVE™from Life Technologies or IFCs from Fluidigm, perform thousands ofnanoliter scale PCR reactions compared to microliter scale typical for96-well or 384-well plates. These platforms typically requirepre-amplification due to low reaction volume. The pre-amplificationreaction is typically diluted 1 to 100× or more and split into N wellsif a single color per target is used or a smaller number of wells ifseveral colors are used for detection in each well.

Also provided by the present invention are ligase-based linkingstrategies. Ligation or ligase chain reaction (“LCR”, see U.S. Pat. No.5,494,810) can be used as a method to add universal segments to one ormore target nucleic acids of interest. Multiplex ligation assays are awell established technique, e.g., SNPLEX™ from Applied Biosystems andGOLDENGATE™ assays from Illumina. One can ligate DNA ligators on bothDNA and RNA templates using appropriate ligases. The ligation reactioncan be temperature cycled using thermostable DNA ligases to achieveamplification for target molecules. For example, see U.S. Pat. No.5,494,810. Ligation assays generally have high specificity for detectionof SNPs and rare mutations because ligases do not ligate nicks between3′ hydroxyl groups and 5′ phosphates of the two ligator oligos, if thereis a mismatch between the 3′ end of the ligator and the target nucleicacid. Similar to the PCR-based pre-amplification described above, theligation reaction can be diluted prior to universal detection.

An example ligase-based linking strategy is schematically illustrated inFIG. 11. As shown, first oligonucleotide 1100 has a 5′ portioncorresponding to first target nucleic acid 1102 and a 3′ portion havingthe universal segment (e.g., universal segment B₁-C₁ in FIG. 11)phosphorylated 5′ end and optionally blocked non-extendable 3′ end.Second oligonucleotide 1104 has a 3′ portion corresponding to firsttarget nucleic acid 1102 and a 5′ portion having the universal segment(e.g., universal segment A₁ in FIG. 11). Ligation of the 3′ end of thesecond universal segment to the 5′ phosphorylated end of the firstuniversal segment links the two universal segments into a singlemolecule in a manner dependent on the first target nucleic acid. Theresulting first tagged target nucleic acid can then be detected usingany of the universal detection formats described herein.

FIG. 12 schematically illustrates a second ligase-based linkingstrategy. As shown, first universal segment 1200 and second universalsegment 1202 are ligated to first target nucleic acid 1204 at ligationpoints 1206 and 1208, respectively. This method can be used when nucleicacids have defined ends, that occur either naturally or artificially,e.g., after using DNA restriction enzymes. The ligation depends on twocomplementary oligos 1214 and 1216 that span the nicks between thetarget nucleic acid and universal tags 1200 and 1202, providing thetemplate for ligation.

It is preferable to remove unligated ligators from the reaction prior touniversal detection. The unligated 5′-P ligator oligos (e.g., 11005′P-target-i-B1-C1-3′ in FIG. 11) can be removed prior to PCR by, e.g.,lambda-exonuclease treatment. This will prevent, e.g., the seconduniversal primer (Ci) in FIG. 1 from priming on these ligators duringuniversal detection. Alternatively, one can perform ligation onsurface-bound target nucleic acid templates and wash off the unligatedligator oligos, e.g., as is done in the GOLDENGATE™ assay from Illumina.

A further universal segment linking strategy, where the universalsegments are linked into a single molecule by reverse transcription(RT), is schematically illustrated in FIG. 13. As shown, first primer1300 has a 3′ portion that anneals to first target RNA 1302 and 5′portion having a first universal segment (A1). Extension of the firstprimer yields first complementary DNA (cDNA) strand 1304. RT extensionsusing primers with target specific 3′-ends, especially when multiplexed,can generate non-specific extensions on the RT primers themselves. Tominimize these non-specific extensions, the Ai tails (1300 in FIG. 13)are optionally blocked by complementary oligos that have blocked 3′ endsor otherwise are non-extendable. Second primer 1306 has a 3′ portionthat anneals to first cDNA strand 1302 and a 5′ portion having a seconduniversal segment (B1-C1). Extension of the second primer yields doublestranded cDNA product 1308, which can be detected using any of theuniversal detection formats described herein.

Linear polymerase extension can also be used to link the first andsecond universal segments together, so long as the target nucleic acidhas a defined 3′ end. An example linear extension linking strategy thatcan be used to detect methylation status is schematically illustrated inFIG. 14. As shown, the sample is divided into two and treated with (a)the methylation-sensitive restriction endonuclease HpaII (which cutsonly unmethylated CCGG sites) and (b) the restriction endonuclease MspI,which cuts all CCGG sites regardless of methylation status. At (c) and(d), a pool of oligos is added to each sample to interrogate specific NCpG sites in the genome, with each oligo having tagged 5′ ends (Aiu in(c), Ait in (d)), and target-specific 3′ ends. A polymerase extends the3′ ends, replacing “CGG” with complements of the Aiu and Ait taggingsequences. The two sub-samples are optionally pooled together (e) plus(f), and multiplex PCR encoding is carried out with preamplificationprimers, e.g., similar to FIG. 15 below. As described elsewhere herein,universal detection measures methylation similar to the singlenucleotide polymorphism detection described at FIG. 15, except real-timePCR is used and deltaCt values between the two colors in each wellindicate the amount of methylated DNA in the sample. Typically, oneneeds to compare deltaCt values in a sample with 0% and 100% methylatedcontrol DNA samples to accurately measure methylation level in samplesof interest.

Another methylation detection method provided by the present inventionuses HpaII or any other methylation-sensitive enzyme or a combination ofenzymes and places target-specific primer with encoding 5′ universalsegments on both sides of the target restriction sites. Generally,multiplex preamplification is performed for multiple methylationtargets. This method, provided the restriction is complete, will measureonly the amount of methylated DNA in the sample and will be able todetect small amounts of methylation in the sample, e.g., abnormallymethylated cancer cells in the background of normal non-methylated DNA.One can multiplex encoding for methylation with CNV, somatic mutation,SNPs and other genomic DNA features of interest in a genomic DNA sample.

Exemplary Applications

The universal detection methods and compositions provided by the presentinvention are applicable for the detection of any type of target nucleicacid of interest. As noted above, exemplary target nucleic acids ornucleic acid features that can be tagged and detected in accordance withthe present invention include DNA and/or RNA (e.g., from primates (e.g.,humans), rodents, viruses, bacteria, Archaea, etc), cDNA, cDNAcorresponding to a short RNA, a genetic variant, a mutation or insertionthat confers drug resistance, a somatic mutation, a polymorphism, asingle nucleotide polymorphism, a rare allele, a portion of the KRASgene, a nucleic acid that exhibits a variation in copy number, anintron, an exon, an intron-exon boundary, a splice junction, one or moredinucleotides corresponding to one or more methylated or unmethylatedCpG dinucleotides in bisulphite treated DNA, a nucleic acid comprisingone or more restriction enzyme recognition sequences, a portion of humanchromosome 21, a portion of the human X chromosome, and a portion of thehuman Y chromosome. It will be understood that any of the universaldetection formats and universal linking strategies (and any combinationthereof) can be used to detect these and other target nucleic acidsand/or nucleic acid features of interest. The exemplary applicationsdescribed herein are merely illustrative and do not serve to limit thetypes of target nucleic acids for which the present invention finds use.

Universal Detection of Polymorphisms and Somatic Mutations

Polymorphism detection is widely used in applications spanning clinicaldiagnostics, human disease research, epidemiology, sub-speciesidentification and tracking, plant and animal breeding, quantitativetrait loci (QTL) mapping, bacterial/viral strain typing, etc.Polymorphisms can be single nucleotide polymorphisms (SNPs), indels,multiple nucleotide polymorphisms (MNPs), inversions, etc.

As an example of how methods and compositions of the present inventioncan be used to detect polymorphisms, a strategy for detecting SNPs isschematically illustrated in FIG. 15. At step (a), three primers per SNP(“i”, where “i”=1 to N) are used for preamplification. Firstallele-specific primer 1500 has a 3′ end specific for allele “x” of SNP“i”, and second allele-specific primer 1502 has a 3′ end specific forallele “o” of SNP “i”. The 5′ portions of the first and secondallele-specific primers include universal segments, where the universalsegments of the allele-specific primers are different. In FIG. 15, the5′ portion of first allele-specific primer 1500 has universal segmentdesignated Aix, and the 5′ portion of second allele-specific primer 1502has universal segment designated Aio. Third common opposite strandprimer 1504 has a locus-specific 3′ end and a 5′ portion having auniversal segment (in this example, 5′-Ci-Bi). The pre-amplification PCRmultiplexes 3*N primers in low concentrations to amplify N SNP loci.Assuming two color detection, the pre-amplification products areoptionally diluted, e.g., 4-64 fold dilution, and split into N universaldetection reactions.

In this example, universal detection is carried out using a formatsimilar to that shown in FIG. 1. For SNP “i”, PCR is performed using twodifferentially labeled primers. Here, D1 and D2 represent fluorescentdyes that can be distinguished from each other, e.g., by havingdifferent emission spectra. As shown at (b), first universal primer 1506includes D1-Bi-Aix-3′ and anneals at portion A′ix of amplicon 1508.Second universal primer 1510 includes D2-Bi-Aio-3′ and anneals atportion A′io of amplicon 1512. Extension of the first and seconduniversal primers generates extension products 1514 and 1516,respectively. The extension products form intramolecular hairpins havingstems between Bi and B′i. Third universal primer 1518 (including portionCi) anneals to portion C′i of extension products 1514 and 1516. 5′nuclease of a polymerase extending from the third universal primerremoves the label D1 and D2 from extension products 1514 and 1516,respectively, generating fluorescent signal corresponding to allele “x”or “o” of the SNP “i” in colors D1 and D2, respectively.

Genotyping read-out can be done as an end-point to detect homozygosityand heterozygosity, or in real-time to accurately measure the ratio ofthe two alleles, e.g., for allele-specific expression or SNPs in copynumber variation (CNV) regions. Polymorphism detection can bemultiplexed, e.g., for two or three SNPs in each universal PCR well,provided the systems used are configured to detect and separate four orsix fluorescent colors, respectively.

Somatic mutations occur, e.g., in cancer, and are often present in avery small portion of the sample DNA. Detection of specific mutations inthe KRAS gene is used to select cancer therapeutics. For example,cetuximab does not work in tumors with certain KRAS mutations. In thiscase, only one “allele-specific” (for the mutation allele) primer can beused (as opposed to two for SNP genotyping), and several mutations canbe multiplexed together based on same or different detection color. Itis important to increase the specificity of allele-specific priming sothat “wild type” predominant alleles from normal cells do not generatefalse positive signal. This application is often referred to as “rareallele detection”. There are several ways to increase the specificity ofpriming for both SNPs and rare allele detection: (a) lowerpre-amplification primer concentrations; (b) shorten the allele-specific3′ ends of the pre-amplification primers, and run the first cycles ofpre-amplification at a lower temperature to engage short primersfollowed by regular cycling when the long tailed primers are engaged;(c) incorporate mismatches in the allele-specific primers close to the3′ end, where the presence of two mismatches has a synergistic effect onprimer specificity; and incorporate modified bases close to the 3′ endsof primers, e.g., as described in U.S. Pat. No. 6,794,142.

Universal Detection for Gene Expression

One of skill will appreciate that the universal detection methods andcompositions of the present invention provide a powerful approach formeasuring gene expression levels, including the simultaneous measurementof the expression levels of many different genes of interest. Anexemplary strategy for simultaneously (e.g., in the same well) measuringthe expression levels of two different genes is schematicallyillustrated in FIG. 16. Preamplification (“encoding”) of cDNA 1600corresponding to mRNA transcribed from a first gene and cDNA 1602corresponding to mRNA transcribed from a second gene occurs at step (a).With respect to cDNA 1600 corresponding to mRNA transcribed from a firstgene of interest, primer 1604 has a 3′ portion that anneals to cDNA 1600and a 5′ portion having first universal segment 5′-A1i. Primer 1606 hasa 3′ portion that anneals to cDNA 1600 and a 5′ portion having seconduniversal segment A1i. Extension of first universal primer 1608 occursat step (b), where first universal primer 1608 has a 3′ portion (A1i)that anneals to portion A′ 1i of the amplicon generated duringpreamplification, and where first universal primer 1608 has a 5′ portionthat includes 5′-D1-B1i-Q. D1 is a first detectable label (e.g., afluorescent dye) and Q is a quencher. At step (c), the product extendedfrom first universal primer 1608 forms an intramolecular hairpin havinga stem between portions B1 i and B′1i. Subsequent to hairpin formation,second universal primer C1i anneals to the extension product and isextended by a polymerase. The 5′ nuclease activity of the polymeraseremoves the first detectable label D1, such that D1 is no longerquenched by Q and emits a first detectable signal (e.g., a color thatcorrelates to the presence or amount of cDNA 1600 in the sample).

Preamplification of cDNA 1602 occurs in a manner similar to that of cDNA1601, but cDNA 1602 is “encoded” during preamplification with universalsegments that are different than those used to encode cDNA 1600. Primer1610 has a 3′ portion that anneals to cDNA 1602 and a 5′ portion havingthird universal segment 5′-A2i. Primer 1612 has a 3′ portion thatanneals to cDNA 1602 and a 5′ portion having fourth universal segment5′-C2 i-B2i. Extension of third universal primer 1614 occurs at step(b), where third universal primer 1614 has a 3′ portion (A2i) thatanneals to portion A′2i of the amplicon generated duringpreamplification, and where third universal primer 1614 has a 5′ portionthat includes 5′-D2-B2i-Q. D2 is a second detectable label (e.g., afluorescent dye) having an emission spectrum distinguishable from D1,and Q is a quencher. At step (c), the product extended from thirduniversal primer 1614 forms an intramolecular hairpin having a stembetween portions B2 i and B′2i. Subsequent to hairpin formation, fourthuniversal primer C2 i anneals to the extension product and is extendedby a polymerase. The 5′ nuclease activity of the polymerase removes thesecond detectable label D2, such that D2 is no longer quenched by Q andemits a second detectable signal (e.g., a color that correlates to thepresence or amount of cDNA 1602 in the sample) that is distinguishablefrom the first detectable signal.

Real-time PCR is frequently used to measure gene expression. The methodsand compositions provided herein can be used to measure expression forseveral splice junctions in a well, each using a different color. Ingene expression applications (e.g., FIG. 16), each target can have twodifferent pre-amplification primers (vs. one common primer forpolymorphisms, methylation, etc.): 5′-A1i-target1-3′,5′-C1i-B1i-target1-3′ and 5′-A2i-target2-3′, 5′-C2i-B2i-target2-3′ (FIG.16( a), assuming 2-color detection). The dye-labeled primers have adifferent 5′ tails, e.g., D1-B1i-Q-A1i-3′ and D2-B2i-Q-A2i-3′ andgenerally two digesting primers can be used: C1 i and C2 i (FIG. 16( b),i=1 to N).

The strategy set forth in FIG. 16 is merely exemplary, and it will beunderstood that any of the universal detection formats and universalsegment linking strategies described herein can be employed inaccordance with the present invention to detect or measure theexpression of one or more genes or any other DNA targets (simultaneouslyor otherwise).

Simultaneous Copy Number Variation and Gene Expression Measurement

Differential gene expression is typically due to transcriptionalregulation, but it is becoming increasingly clear that copy numbervariations (CNVs) are frequent in humans and contribute to thedifferential expression level of genes that are located in chromosomalregions that exhibit variations in copy number. The universal detectionmethods and compositions of the present invention are well-suited tomeasure CNVs, much in the same way that gene expression is measured, butwhere the target nucleic acid of interest is a portion of a gene (e.g.,an intronic region, a region spanning an intron-exon boundary, etc.),rather than a cDNA corresponding to an mRNA transcribed from a gene.

Copy number variation can be measured alone, or can be measuredsimultaneously with the detection of other target nucleic acids thatprovide additional useful information, e.g., gene expression levels,methylation status, chromosomal abnormalities, and the like. An exampleapplication for the simultaneous measurement of copy number variationand gene expression is schematically illustrated in FIG. 17. To measurethe number of copies of a target gene of interest in a sample (relativeto a control sample), a preamplification reaction using firstpreamplification primer 1700 and second preamplification primer 1702 isperformed at step (a). First preamplification primer 1700 has a 3′portion that anneals to an intron of gene 1704, and a 5′ portion havingfirst universal segment 5′-Ai. Second preamplification primer 1702 has a3′ portion that anneals to an exon of gene 1704, and a 5′ portion havingsecond universal segment 5′-Ci-Bi. At step (b), a 3′ portion of firstuniversal primer 1706 anneals to the amplicon generated duringpreamplification. First universal primer 1706 has a 5′ portion thatincludes 5′-D1-Bi-Q, where D1 is a first detectable label (e.g., afluorescent dye) and Q is a label quencher. First universal primer 1706is subsequently extended by a polymerase, and at step (c), the extensionproduct forms an intramolecular hairpin having a stem between segmentsBi and B′i. Following hairpin formation, second universal primer 1708(including segment Ci), anneals to the extension product and is extendedby a polymerase. The 5′ nuclease activity of the polymerase removes thefirst detectable label from the extension product, resulting in a firstdetectable signal from D1.

Simultaneous measurement of gene expression is shown at the right ofFIG. 17. cDNA 1712 corresponding to an mRNA transcribed from gene 1704is amplified using third preamplification primer 1710 and secondpreamplification primer 1702. Detection is initiated at step (b) byextension of third universal primer 1714 having a 3′ portion thatanneals to the preamplification amplicon and a 5′ portion that includes5′-D2-Bi-Q, where D2 is a second detectable label (distinguishable fromD1) and Q is a label quencher. Third universal primer 1714 issubsequently extended by a polymerase, and at step (c), the extensionproduct forms an intramolecular hairpin having a stem between segmentsBi and B′i. Following hairpin formation, second universal primer 1708anneals to the extension product and is extended by a polymerase. The 5′nuclease activity of the polymerase removes the second detectable labelfrom the extension product, resulting in a second detectable signal fromD2 that is distinguishable from the signal generated by D1. The amountof D1 signal (e.g., Ct-value for the first fluorescent color) correlatesto gene copy number, while the presence or amount of D2 signal (e.g., asecond fluorescent color) correlates to gene expression level. Thesecond color D2 can be used to measure another genomic locus as acontrol. For example, the RNAse P gene locus always exists as two copiesin a diploid genome, and can serve as a control that can be measured inthe same well using color D2, while the target CNV is measured usingcolor D1. In this case, CNV detection will be similar to cDNA detectionas shown in FIG. 16, but using genomic DNA (gDNA) targets rather thancDNA targets. The deltaCt value between D1 and D2 in the same wellcorrects for pipetting errors and allows more accurate measurement ofCNVs.

Two-Strand Universal Detection

The universal detection methods and compositions provided by the presentinvention can be used for two-strand detection of a target nucleic ofinterest. An exemplary approach for two-strand detection isschematically illustrated in FIG. 18. As shown, first tagged targetnucleic acid 1800 includes first universal segment 1802 and seconduniversal segment 1804. Both the first and second universal segmentsinclude two sub-segments: first universal segment 1802 includes Ai-Ei(and A′I-E′i), and second universal segment 1804 includes B′i-C′i (andBi-Ci). First tagged target nucleic acid 1800 is denatured, and upon asubsequent decrease in temperature, first universal primer 1806 annealsto a first end of one strand of target nucleic acid 1800, and seconduniversal primer 1808 anneals to a second end of the same strand oftarget nucleic acid 1800. First universal primer 1806 has a 3′ portionthat anneals to first universal segment 1802 and a 5′ portion thatincludes 5′-D1-Bi-Q. Second universal primer 1808 has a 3′ portion thatanneals to second universal segment 1804 and a 5′ portion that includes5′-D2-Ei-Q. D1 and D2 are detectable labels that are distinguishablefrom each other, and Q is a label quencher. The first and seconduniversal primers are extended to generate the extension products shownat (c) and (e), respectively. At (c), the extension product from firstuniversal primer 1806 forms an intramolecular hairpin having a stembetween Bi and B′i. Subsequent annealing and extension of seconduniversal primer 1808 removes label D1 from the extension product,resulting in a detectable signal from D1. At (e), the extension productfrom second universal primer 1808 forms an intramolecular hairpin havinga stem between Ei and E′i. Subsequent annealing and extension of firstuniversal primer 1806 removes label D2 from the extension product,resulting in a detectable signal from D2 that is distinguishable fromthe signal generated by D1.

Universal Detection of Methylation Status

The present invention also finds use in detecting epigenetic features,e.g., features affecting the structure of genomic DNA which are notdirectly related to the primary DNA sequence. Methylation of genomic DNAand histones are common epigenetic features that affect genomic DNAstructure and transcriptional regulation. Methylation of CpGdinucleotides in genomic DNA (met-C) is known to correlate withtranscriptional regulation. For example, cancer cells often haveabnormally methylated regions in the vicinity of transcription startpoints for tumor suppressor genes, causing down regulation of theirtranscription. Bisulphite converted DNA is frequently used to measuremethylation levels in genomic DNA. Bisulphite treatment converts allunmethylated cytosines (C) into uracil (U) residues that behave asthymidines (T) during PCR. Bisulphite treatment does not convertmethylated cytosines to uracils. As such, methylation detectionaccording to the present invention can be performed in a manner similarto that described for SNP genotyping: if a given locus is detected as“CG”, then that cytosine in the genomic DNA was methylated; if the locusis detected as “UG”, then the cytosine was not methylated.

An example approach for detecting the methylation status of a genomiclocus of interest using the universal methods and compositions of thepresent invention, is schematically illustrated in FIG. 19. In thisexample, the methylation status of two closely-spaced CpG dinucleotidesis performed using two-strand detection in two/four colors. Both strandsare shown as they exist after bisulphite conversion; dsDNA is shown,although the sequences will actually be single-stranded.Pre-amplification encoding at (a) uses two met-C specific primers 1902and 1904, but four primers for sites converted from C to U: two with aCA-3′ end (1906 and 1908) that start at the first cycle and two with aT-3′ end (1910 and 1912) that engage starting at the second cycle.Alternatively, one of the pairs, e.g., 1906 and 1910 can be used todetect one of the methylated strands. Molecules with only one methylatedsite are shown at (c) 1914 and (d) 1916. A total of N sites can bemultiplexed during preamplification/encoding. The methylation status isencoded by the 5′ tails: Aim and Cim for methylated DNA and Aiu and Ciufor unmethylated DNA. Two middle tags, Ei and Bi, encode two CpG sites.Four universal detection primers are used for the detection in fourcolors for each pair of methylation sites. the primers containingD1-Bi-Q-Aim-3′ and D2-Ei-Cim-3′ detect methylated DNA in colors D1 andD2 for the CpG sites shown at the left and right, respectively. Theprimers containing D3-Bi-Q-Aiu-3′ and D4-Ei-Ciu-3′ detect unmethylatedDNA. The ratio of signals D1/D3 and D2/D4 correlate with percentagemethylation for the left and right CpG, respectively.

Universal Detection for Measuring Short RNA Expression

Short RNAs present a challenge for TAQMAN™ detection as the length ofthe target RNA is not long enough for both primers and probes toanneal/hybridize. The universal methods and compositions of the presentinvention can be readily employed to detect such short RNA sequences. Anexample universal segment linking strategy (“encoding”) method inaccordance with the present invention for measuring expression levels ofshort RNAs, e.g., mature miRNAs, is schematically illustrated in FIG.20. A reverse transcription (RT) step uses multiplexed RT primers (asshown, RT primer 2000 and RT primer 2002) with 3′ ends matching the 3′ends of miRNAs and 5′ universal segments tagging each miRNA sequence.These RT primers predominantly reverse transcribe mature miRNAs becausethe miRNA precursors are normally folded into stable hairpins. One canoptionally add “blocking oligos” complementary to the universal 5′ tailsof the RT primers to minimize RT primers priming on other RT primers.The next step is linear pre-amplification (or alternatively, PCR) usingforward primers (as shown, primer 2004 or primer 2006) with 5′ universalsegments and 3′ ends specific to 5′ ends of miRNAs (and optionally,primers 2008 or 2010 that match the 5′ universal segment of the RTprimers). Forward primers 2004 and 2006 may include one to threenon-template Gs (guanosines) between the target-specific region anduniversal 5′ tails, to stabilize forward primer annealing to thenon-template Cs (cytosines) that RT often adds at the ends of RNA. Anyof the universal detection formats described herein (e.g., the universaldetection format illustrated in FIG. 1) can be used, with one or morecolors per well based on how many miRNA species are multiplexed in awell.

Universal Detection of Drug Resistance Mutations

More accurate and powerful approaches for detecting mutations thatconfer drug resistance would greatly facilitate the individualizedtreatment of individuals affected by diseases, e.g., for which more thanone treatment option is available. As will be appreciated, the universaldetection methods and compositions provided by the present inventionconstitute a significant advancement in the field of personalizedmedicine.

The treatment of individuals infected with human immunodeficiency virus(HIV) typically includes a combination of three or more drugs. Duringthe course of treatment, resistance to these drugs may develop, and itis advantageous to promptly replace the drug to which the HIV hasdeveloped resistance with another drug. The standard test used todayinvolves Sanger sequencing of regions in the reverse transcriptase (RT)and protease inhibitor (PI) genes. This method is relatively expensive,takes a long time (10 days on average in a service lab) and can detectmutations only if they comprise >20% of total viral load. As provided bythe present invention, known mutations that confer resistance toantiretroviral drugs administered in a drug cocktail can be encoded anddetected in several hours in such a way that any resistance mutation toeach drug is indicated by its own color, and a small percentage ofmutated viruses in the viral population can be detected. Detailed andcurrent information regarding mutations that confer resistance toantiretroviral drugs can be found at Stanford University's HIV DrugResistance Database.

An example of how the present invention can be used for multiplexmutation detection for HIV drug resistance is schematically illustratedin FIG. 21. Mutations in HIV protease that confer resistance to threeprotease inhibitors (ATV, DRV and FPV) are shown at the top of FIG. 21.Shown at (a) are mutation-encoding (preamplification) primers 2100-2106for positions 24-54 and one locus-specific primer 2108 that includesC-B1-target-3′. For positions 73-90, shown at FIG. 21( b),mutation-encoding (preamplification) primers 2110-2116 andlocus-specific primer 2118 that includes C-B2-target-3′ are used. “X”designates the mutation-specific bases in viruses that are detected bycomplementary bases at the 3′ ends of encoding primers; each mutation ingeneral having its own primer with a mutation-specific 3′ end. Shown inFIG. 21( c) are eight labeled universal detection primers 2120-2134 anduniversal primer 2136 (including segment C), which detect mutations suchthat drug resistance is indicated in one of the four colors: ATVresistance is indicated by D1; ATV and FPV resistance is indicated byD2; DVR and FPV resistance is indicated by D3; and resistance to allthree drugs is indicated by D4. In addition, total viral load should beencoded by D5 and internal positive control (IPC) as D6 (not shown).FIG. 21 illustrates a general approach for encoding multiple targets (inthis case different mutations that cause resistance to the same drug) tobe detected in the same color. One can simplify this example by usingsame tags B1 and B2 and same A_(j1) and A_(j2) (j=1 to 4), so that fourrather than eight labeled primers will be required for detection. Onecan also use the “switched format” detection method with a 5′ quencherdescribed above: when a color is detected during qPCR indicating thatvirus has developed a resistance, one can size fluorescent PCR productsby capillary electrophoresis and determine which amino acid residue ismutated based on the length of the PCR product.

As an example, FIG. 21( a) shows encoding primer 2100 havingA11-target-G48V-3′ or A11-target-G48M-3′ (top left corner) with a 3′-endthat matches mutations that change the wild type 48G (glycine) to valineor methionine. These primers do not prime on the nucleic acid encodingthe wild type 48G in HIV protease. This test will be simpler to use thanthe PCR Sanger sequencing test currently in use and will be able todetect less than 20% viral subpopulations that are not detectable bySanger sequencing. For example, a GENEXPERT™ system from Cepheid candetect 6 colors in a cartridge, enabling the detection of resistance tothree antiretroviral drugs as well as total viral load in a singlecartridge in several hours. When mutated viruses are present, the testwill indicate if resistance to each drug in a cocktail has emerged.Physicians can then modify the treatment cocktail/regimen accordingly.

The most frequently used HIV combination therapies (or “drug cocktails”)have two nucleoside RT inhibitors (NRTI) and one non-nucleoside RT(NNRTI) or protease inhibitor (PI). Accordingly, four colors will besufficient to detect four possible combinations of resistance to eachdrug and two NRTIs together. Detection colors are selected in such a waythat each drug has a different detection color for all mutations thatcause drug resistance. Several drugs used in a drug cocktail orcandidates for treatment can be tested in multiplex using availablecolors in the detection instrument. One color can be used to measuretotal viral load.

High-Throughout Universal Detection

Exemplary Implementation on Integrated Fluidics Chips

An example implementation of the universal detection methods andcompositions of the present invention on integrated fluidics chips(“IFCs”, e.g., those commercially available from Fluidigm) isschematically illustrated in FIG. 22. This example assumes four-colordetection, although any number of colors detectable by the instrumentcan be used. The IFC has N sample and M assay loading wells so that thetotal number of detectable targets is 4*N*M. Currently available IFCsare N=M=48 (48×48) and N=M=96 (96×96). If the goal, for example, is tomeasure 8*M targets in a sample, two pre-amplification PCRs, each for4*M targets each are performed for N/2 samples and PCR pre-amplificationproducts for each sample are loaded into two sample-loading wells. Fourpreamplification primer sets 2200-2206 are shown at FIG. 22( a). A totalof 4*M primers are used in each pre-amplification PCR. M universaldetection primer sets 2208-2214, four primers each, FIG. 22( b) areloaded into M orthogonal loading wells (“i”, where i=1 to M). Universaldetection primer set 2208 detects nucleic acids tagged usingpreamplification primer set 2200, and so forth. N pre-amplificationreactions are mixed with M universal detection assays and 4*N*M targetsare measured using real-time (quantitative) PCR and/or end-point (e.g.,“digital”) PCR in N*M wells (FIG. 22( c)).

The 8*M targets used in this example (FIG. 22) can be any combination ofSNPs, somatic mutations, CNVs, translocations, etc., all multiplexedtogether from the same gDNA sample. Gene expression, miRNA and/ormethylation targets can also be included among the 8*M targets usingseparate sample preps and shared or additional pre-amplifications.

Digital PCR

Digital PCR is a method to accurately count the number of DNA moleculesin a sample. For example, see U.S. Pat. No. 6,143,496. The methodrequires a small number of target DNA molecules and a large number ofwells or droplets, the latter generally higher than the first. Assumingsingle-molecule PCR sensitivity and specificity, one can count thenumber of negative and positive wells/droplets and assuming randomdistribution to accurately count the initial number of target moleculesin the sample. When using approaches provided by the present inventionfor digital PCR, the number of pre-amplification cycles andpost-amplification dilution are adjusted such that the number of targetmolecules is smaller or similar to the number of digital PCR mini-wellsor droplets. As provided by the present invention, digital PCR can beused to count any nucleic acid target, e.g., genomic DNA targets,methylated DNA, mRNA/miRNA/ncRNA, viruses, mutated viruses, etc.

Methods known in the art can be used to deliver universally taggedtarget nucleic acids (e.g., pre-amplified DNA) to multiple distinctdigital count detection containers, e.g.: (1) traditional96-384-1,536-3,072 and so on well micro-titer plates; (2) Fluidigm andBioTrove use thousands of mini-wells in specialized nano-fluidicsdevices; (3) systems from 454 Life Sciences/Roche use a PICOTITERPLATE™device with more than a million wells; or (4) small droplets can be madeby emulsion PCR (ePCR, currently used commercially by 454/Roche andSOLID™/Life technologies) or specialized devices to make small waterdroplets in oil, e.g., RainDance RAINSTORM™ or QUANTALIFE™. Digitalcounts of positive droplets (e.g., droplets positive for detectablesignal generated by the detection steps of the universal detectionmethods of the present invention) and negative droplets can be countedusing a counting device.

FIG. 23 schematically illustrates an example digital PCR method fordiagnosing trisomy 21 (T21) in DNA from mother's blood using anintegrated fluidics chip in accordance with the present invention. Asshown at (a), 48+48 unique targets are selected from chromosome 21, 48are selected from other autosomes, and 48 are selected from chromosome Xfor a total of 192 loci. A single 192-plex preamplification (“encoding”)PCR is diluted, e.g., to approximately 400-1,000 molecules for eachchromosome and mixed with four universal detection primer sets 2300-2306(shown at FIG. 23( b)) that anneal to the universal segmentscorresponding to the Down syndrome critical region (DSCR) on chromosome21 (2300), autosomes (2302), the X chromosome (2304) and remainingportions of chromosome 21 (2306). As shown at (c), each universaldetection reaction is loaded onto one of 12 loading wells (largecircles) and split into 1,000 mini-wells or droplets (small circles indashed line) for universal detection using the methods and reactionmixtures of the present invention. 48,000 dPCR data points (12*1,000*4colors) will permit an accurate calculation of the number of taggedtarget nucleic acid molecules for chromosome 21, autosomes andchromosome X, based upon the number of positive and negative wells ordroplets in each color.

One can perform digital count for T21 detection using universal ePCR(FIG. 7). The beads for ePCR have four universal detection primers:Q-B1-D1-spacer-surface-spacer-A1-3′,Q-B2-D2-spacer-surface-spacer-A2-3′, Q-B3-D3-spacer-surface-spacer-A3-3′and Q-B4-D4-spacer-surface-spacer-A4-3′ (e.g., FIG. 7( a) shows two suchprimers). The pre-amplification products and the universal “C” primer(e.g., see FIG. 7( b)) are used in the ePCR with the four-primer beads,so that each droplet has on average less than one pre-amplified targetin each color. During ePCR several tens of thousands of 5′nuclease-specific fluorescent dye molecules will be unquenched on eachbead as result of the exponential amplification starting from a singlepre-amplified molecule (FIG. 7( c)-(d)). Next, the ePCR emulsion isbroken and the beads are deposited on the slide surface. The read-outhas several steps: (1) the total number of beads is counted in whitelight; and (2) the fluorescent detection is performed in four colors,with each bead can be positive for as many as four colors. Given theimportance of avoiding false negatives, two optional additional controlssteps can be performed: one or several cycles of hybridization using aset of labeled in four color probes that are specific to each of thetargets used for detection (this orthogonal direct target detection isused to confirm the sequence that gave rise to the universal detectionsignal on each bead; and treatment of the slide with reagents that cutthe bond between the quencher from the dye, so that every bead showssignal in four colors. This can be done, e.g., if uridine, THF, or oxo-Gis present between the quencher and the dye and the enzymatic treatmentwith UDG/EndoV/Fpg, respectively, is used to cleave off the quencherfrom all beads. The total unquenched signal effectively estimates thenumber of universal primers bound to each bead during manufacturing.This signal can be compared to the universal detection signal to betterdistinguish between real and false positive signal on each bead.Essentially, we normalize signal per bead given that during beadmanufacturing, a different number of universal primer molecules can beattached to each bead.

The number of the pre-amplification molecules for chr21, autosomes and Xcan be compared. Given that each slide can have hundreds of millions ofbeads, a small 2.5% increase in chromosome 21 counts relative to otherautosomal and X chromosome counts can be detected and used to diagnosetrisomy 21. The same test will also detect X-chromosome aneuploidy,e.g., triple-X, XXY, and/or XO.

The universality of the detection primers of the present invention makesit much easier to develop detection on the beads or on the surfaces: thesame beads or slide surfaces can be used to detect any set of targets.But in cases where a very large number of samples needs to be tested,e.g., for detection of trisomy 21, one can use target-specific labeledprimers (e.g., as in FIGS. 7-9), on the beads or on the slide surfacesto directly measure nucleic acids or use non-tailed primers topre-amplify genomic DNA/RNA.

A second approach provided by the present invention for diagnosingtrisomy 21 via digital PCR is schematically illustrated in FIG. 24.First, one selects short B1 sequences that occur more than 48 times inthe Down syndrome critical region (DSCR) on chromosome 21, anothersequence B2—in autosomes, B3—in chromosome X and B4 in remainingportions of chromosome 21. 48 encoding primer pairs with 5′ universalsegments (“tails”) A1/C1 are designed to amplify 48 B1 loci in DSCR.96*3=288 encoding primer pairs with tails A2/C2, A3/C3 and A4/C4 aredesigned to amplify loci that with sequences B2, B3 and B4 in respectivechromosomes. As shown at (b) universal primers to detect tagged targetnucleic acids from DSCR have short 5′-tail B1 labeled in D1 (universalprimer 2400); autosomal targets have tail B2/D2 (universal primer 2402),chromosome X targets have tail B3/D3 (universal primer 2404), and therest of chromosome 21 having tail B4/D4 (universal primer 2406). Asingle 48+288=336-plex, or alternatively, 12×28-plex or any combinationin-between of multiplex encoding PCR reactions are performed (notshown). Twelve preamplified (“encoded”) samples are diluted toapproximately 4,000-8,000 molecules for each target and mixed with thefour labeled universal detection primers 2400-2406 and correspondingreverse universal detection primers 2408-2414 (b) that target DSCR,autosomes, the X chromosome and the rest of chromosome 21. As shown at(c), each universal detection reaction is loaded into one of 12 loadingwells (large circles) and split into 10,000 mini-wells or droplets(small circles). 480,000 dPCR data points (12*10,000*4 colors) permitaccurate calculation of the number encoded molecules for chromosome 21,autosomes and the X chromosome based upon the number of positive andnegative wells or droplets. It will be understood that the presentinvention contemplates any number of sample loading wells, assay loadingwells, and reaction locations (e.g., detection wells) For example, 1,000or 10,000 wells or droplets, 12 loading wells, 48 or 96 targets forDSCR, X, autosomes, etc. can be used.

Other prenatal and clinical testing can include CNVs, translocations,etc. As personalized genomics and pharmacogenomics applications expand,SNP, CNV, gene expression, miRNA, methylation and rare allele detectionvia the methods and compositions provided by the present invention canbe used for both clinical and research use.

One possible application is cancer diagnosis and/or cancerpharmacogenomics, but there are other clinical conditions where thisapproach can be used. For example, one can detect multiple viral and orbacterial pathogens in a sample. Cancer is linked to many underlyinggenomic, epigenomic and gene regulation changes. SNPs and otherinherited (germ line) polymorphisms can predispose to cancer, e.g.,BRCA1/2 gene alleles. In addition, somatic mutations, large genomicrearrangements, e.g., translocations (BCR-ABL is the most well-known),CNVs (including loss or heterozygocity) and other changes in genomic DNAare linked to cancer. It is well established that cancer andpre-cancerous cells have different gene and miRNA expression patterns ascompared to normal cells. Patterns of differential gene expression areused to differentiate between different types of leukemias. For example,Genomic Health Inc. has commercialized the ONCOTYPEDX™ test to measureexpression levels for 21 genes and recommend chemotherapy for breastcancer patients based on the results. Cancer has many different causesand a large number of available chemotherapeutic drugs either work or donot work depending on the underlying genetic abnormalities (somaticmutations, methylation, gene regulation, etc.).

FIG. 25 schematically illustrates an example diagnostic applicationusing the universal detection methods and compositions of the presentinvention. Here, an integrated fluidic chip (IFC, e.g., from Fluidigm)is used, and SNPs and/or somatic mutations (FIG. 25( a), mRNA/miRNAexpression (FIG. 25( b)) and methylation (FIG. 25( c)) are encoded bypreamplification PCR which links universal segments for each particulartarget. The target nucleic acid in each of the N sample-loading wellshas to be encoded by 4*M encoding primers; only encoding primers thatare decoded by A_(1i), B_(1i) and C_(1i) in color D1 are shown. Theencoding primers with tails 5′-A_(2i)/5′-C_(2i)-B_(2i),5′-A_(3i)/5′-C_(3i)-B_(3i) and 5′-A_(4i)/5′-C_(4i)-B_(4i); are notshown. As shown at FIG. 25( a), preamplification (“encoding”) primers2500 and 2502 have 3′ portions specific to one or more genomic DNAtargets, e.g., polymorphisms (e.g., SNPs), somatic mutations, CNVs,translocations, and the like. Preamplification primers 2504 and 2506have 3′ portions specific to a cDNA corresponding to an mRNA, an miRNA,an ncRNA, and the like. Preamplification primers 2508 and 2510 have 3′portions specific to a genomic DNA target containing one or more CpGsites for which the methylation status will be interrogated. The 5′portions of preamplification primers 2500-2510 include universalsegments that encode the desired genomic target for subsequent detectionusing the corresponding universal detection primer pairs shown at FIG.25( d).

The tagged target nucleic acids from the linking/encoding step at FIG.25( a)-(c) are detected using the four universal primer pairs 2512-2518shown at FIG. 25( d). Each of the four universal primer pairs aredifferentially labeled and have 3′ portions specific to thecorresponding universal segments linked to the nucleic acid targets at(a)-(c). The tagged (“encoded”) target nucleic acids and the universalprimers are delivered to reaction locations on the IFC (shown at FIG.25( e)) where universal detection occurs. It will be understood thatIFCs can be preloaded with the universal detection primers. For example,IFCs can be sold with the universal detection primers already present inassay loading wells or the reaction locations, and the end-user needonly load the tagged target nucleic acids into the IFC before universaldetection can be carried out in accordance with the present invention.The results for each sample can be combined together into a diagnosticoutput, e.g., a recommended treatment.

As shown in FIG. 25, methods and compositions of the present inventionmethod can be employed to detect any combination of multiple genomic,RNA, methylation targets, and the like, on an IFC. Using four colors, atotal of up to 36,864 targets and controls can be measured in a 96×96IFC. A wide range of samples can be used, e.g., 96 cells from the sameindividual, several tumor and normal tissue control samples for a fewpatient or 96 different patient samples. The data from all targets arethen combined for each patient and used to diagnose or selectappropriate therapy for each individual.

Universal detection assays and/or encoding reagents (e.g.,pre-amplification primers) can be delivered to customers in a variety ofways. The sets of universal UniTaq primers can be preloaded on plates orfluidics devices, like Fluidigm IFCs, BioTrove OPENARRAYS™, IdahoFILMARRAY™, or Cepheid GENEXPERT™ cartridges. These sets can also besold as kits in regular 96/384/1,536-well plates or “pre-plated” inmultiple plates, so that customers only need to add tagged (e.g., bypreamplification or any other linking strategy provided herein) samplesto these plates.

The pre-amplification encoding primers with 5′ universal segmentscorresponding to the universal segments of the universal detectionprimers can be ordered through a Web portal. The pre-amplificationassays can be delivered to customers pooled, e.g., 96+192=288 primersmixed together to detect 96 SNPs. Alternatively, the pre-amplificationprimers can be delivered in plates, e.g., three oligos per well in a96-well plate. The latter gives customers an additional flexibility tochange the list of targets, but requires an additional step of poolingof all pre-amplification primers together. In some researchapplications, the same set of genes/SNPs/miRNAs is measured by differentcustomers. In such cases, pre-amplification primers can be inventoried.

In clinical and non-research applications, pooled pre-amplificationencoding primers would be particularly convenient, as all customers needto detect the same set of targets. Several pools may be bundledtogether, e.g., 192 SNPs detected in two colors may require two 96-SNPpre-amplification primer pools. The bundled pools may include differenttypes of pre-amplification primers, e.g., 96 SNPs in pool one, two poolsfor 192 gene expression targets, one pool for 96 methylation targets,etc. These bundled pre-amplification pools can be used for clinicalapplications when it is desirable to measure different types of DNA(SNP, CNV, mutations, methylation, etc.) and/or RNA markers for eachsample.

Example Universal Detection Composition

As noted herein, the present invention provides compositions foruniversally detecting target nucleic acids of interest. For example, asschematically illustrated in FIG. 26, the present invention providesnucleic acid detection reaction mixtures. As shown, the reactionmixtures includes analyte nucleic acid 2600 having nucleic acidsubsequence of interest 2602. The analyte nucleic acid also includesfirst tag sequence 2604, second tag sequence 2606 and third tag sequence2608. As shown, the second tag sequence is located between the first andthird tag sequences. The reaction mixture further includes firstuniversal primer 2610 that includes first tag complement subsequence2612 that is complementary to first tag sequence 2604. The firstuniversal primer also includes a subsequence that includes second tagsequence 2606 or a subsequence thereof, and detectable label 2614.Optionally, the first universal primer includes label quencher 2616. Thereaction mixture further includes second universal primer 2618 thatincludes the third tag sequence or a subsequence thereof. As usedherein, “including the first tag sequence”, “including the second tagsequence”, and “including the third tag sequence” means that the firstor second universal primer includes a subsequence that is sufficientlysimilar to the first, second, or third tag sequences such that theuniversal primer subsequence is capable of annealing to a complement ofthe first, second, or third tag sequence under the desired reactionconditions (e.g., desired temperature, etc.). Further, it will beunderstood that “tag sequence” includes either strand of the first,second and third tag sequences.

Methods for detecting an analyte nucleic acid using the reaction mixtureas shown in FIG. 26 are also provided by the present invention. Themethods include providing the reaction mixture shown in FIG. 26. One ormore PCR cycles are performed. During PCR, tag complement subsequence2612 of first universal primer 2610 anneals to first tag 2604 of theanalyte nucleic acid, and second universal primer 2618 anneals to thirdtag sequence 2608. The one or more PCR cycles generate product 2620.Product 2620 is melted and the reaction temperature is subsequentlyreduced, such that second tag sequence 2606 of upper strand 2622 ofproduct 2620 forms hairpin stem 2624 with the complementary strand ofsecond tag sequence 2606. Following hairpin formation, second universalprimer anneals to upper strand 2622 and is extended by a polymerasehaving 5′ exonuclease activity. Extension of the second universal primerreleases label 2614 from upper strand 2622, permitting detection of thelabel (e.g., unquenched or reduced quenched label 2614), which detectionindicates the presence or amount of the target nucleic acid. As will beappreciated and described elsewhere herein, the example reactionmixtures and methods illustrated in FIG. 26 can be used (or modified foruse) with any of the example universal detection formats, exampleuniversal segment linking strategies, and exemplary applications(optionally in high-throughput) provided by the present invention.

Target Nucleic Acid Sources and Molecular Biology Reagents andTechniques

As will be appreciated, target nucleic acids that find use in theinvention can be obtained from a wide variety of sources. For example,target nucleic acids can be obtained from biological or laboratorysamples including cells, tissues, lysates, and the like. In certainaspects, the source of target nucleic acids includes cells or tissuesfrom an individual with a disease, e.g., cancer or any other disease ofparticular interest to the user.

A plethora of kits are commercially available for the purification oftarget nucleic acids from cells or tissues, if desired (see, e.g.,EASYPREP™, FLEXIPREP™, both from Pharmacia Biotech; STRATACLEAN™ fromStratagene; QIAPREP™ from Qiagen). In addition, essentially any targetnucleic acid can be custom or standard ordered from any of a variety ofcommercial sources.

General texts which describe molecular biological techniques for theisolation and manipulation of nucleic acids include Berger and Kimmel,Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al.,Molecular Cloning: A Laboratory Manual (3rd Ed.), Vol. 1-3, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 2001 (“Sambrook”) andCurrent Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., (supplemented through the currentdate) (“Ausubel”)).

Labeling strategies for labeling nucleic acids and correspondingdetection strategies can be found, e.g., in Haugland (1996) Handbook ofFluorescent Probes and Research Chemicals Sixth Edition by MolecularProbes, Inc. (Eugene Oreg.); or Haugland (2001) Handbook of FluorescentProbes and Research Chemicals Eighth Edition by Molecular Probes, Inc.(Eugene Oreg.) (Available on CD ROM).

A number of embodiments of the present invention utilize the principlesof polymerase chain reaction (PCR). PCR methods and reagents, as well asoptimization of PCR reaction conditions (e.g., annealing temperatures,extension times, buffer components, metal cofactor concentrations, etc.)are well known in the art. Details regarding PCR and its uses aredescribed, e.g., in Van Pelt-Verkuil et al. (2010) Principles andTechnical Aspects of PCR Amplification Springer; 1st Edition ISBN-10:9048175798, ISBN-13: 978-9048175796; Bustin (Ed) (2009) The PCRRevolution: Basic Technologies and Applications Cambridge UniversityPress; 1st edition ISBN-10: 0521882311, ISBN-13: 978-0521882316; PCRProtocols: A Guide to Methods and Applications (Innis et al. eds)Academic Press Inc. San Diego, Calif. (1990) (Innis); Chen et al. (ed)PCR Cloning Protocols, Second Edition (Methods in Molecular Biology,Volume 192) Humana Press; and in Viljoen et al. (2005) MolecularDiagnostic PCR Handbook Springer, ISBN 1402034032.

As noted herein, the universal detection steps of the present inventioncan be performed in real-time, e.g., where one or more detectablesignals (if any) corresponding to the presence or amount of one or moretarget nucleic acids are detected at the conclusion of one or more PCRcycles prior to completion of thermal cycling. Real-time/quantitativePCR techniques are known in the art. Detailed guidance can be found in,e.g., Clementi M. et al (1993) PCR Methods Appl, 2:191-196; Freeman W.M. et al (1999) Biotechniques, 26:112-122, 124-125; Lutfalla G. and UzeG. (2006) Methods Enzymol, 410: 386-400; Diviacco S. et al (1992) Gene,122: 313-320 Gu Z. et al (2003)/Clin. Microbiol, 41: 4636-4641.Real-time (e.g., quantitative) PCR detection chemistries are also knownand have been reviewed in, e.g. Mackay J., Landt O. (2007) Methods Mol.Biol, 353: 237-262; Didenko V. V. (2001) BioTechniques, 31, 1106-1121;and Mackay L M. et al (2002) Nucleic Acids Res., 30: 1292-1305, whichare incorporated herein by reference in their entireties for allpurposes.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovecan be used in various combinations. All publications, patents, patentapplications, and/or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application,and/or other document were individually indicated to be incorporated byreference for all purposes.

What is claimed is:
 1. A method of detecting the presence or amount of afirst target nucleic acid in a sample, the method comprising: (a)linking first and second universal DNA segments into a first molecule ina linking reaction dependent on the first target nucleic acid, therebyproviding a first tagged target nucleic acid; and (b) PCR amplifying thefirst tagged target nucleic acid using first and second universalprimers, each universal primer having a 3′ portion that anneals to oneof the two universal DNA segments; wherein: the 3′ portion of the firstuniversal primer anneals to the first universal segment; a 5′ portion ofthe first universal primer comprises a nucleic acid sequencesufficiently similar to a portion of the first universal DNA segment, aportion of the second universal DNA segment, or a portion of the firsttarget nucleic acid, to anneal to a complement of the portion of thefirst universal DNA segment, a complement of the portion of the seconduniversal DNA segment, or a complement of the portion of the firsttarget nucleic acid, respectively, under PCR reaction conditions; anamplicon generated upon extension of the first universal primer forms anintramolecular hairpin stem between the 5′ portion of the firstuniversal primer and a portion of the amplicon complementary to thefirst universal DNA segment, the second universal DNA segment, or thefirst target nucleic acid; or wherein the first universal primer forms acircular structure upon annealing to the first tagged nucleic acid thatbrings the 3′ and 5′-ends of the first universal primer close to eachother; and, formation of the hairpin stem or circular structure resultsor causes a change in a first detectable signal, the first detectablesignal indicating the presence or quantity of the first target nucleicacid in the sample.
 2. The method of claim 1, wherein the first targetnucleic acid comprises a nucleic acid or nucleic acid feature selectedfrom: a DNA, an RNA, a bisulphite treated DNA, a mammalian nucleic acid,a primate nucleic acid, a rodent nucleic acid, a viral nucleic acid, abacterial nucleic acid, an archaea nucleic acid, a cDNA, a cDNAcorresponding to a short RNA, a genetic variant, a mutation or insertionthat confers drug resistance, a somatic mutation, a polymorphism, asingle nucleotide polymorphism, a rare allele, a portion of the KRASgene, a nucleic acid that exhibits a variation in copy number, anintron, an exon, an intron-exon boundary, a splice junction, one or moredinucleotides corresponding to one or more methylated or unmethylatedCpG dinucleotides, a nucleic acid comprising one or more restrictionenzyme recognition sequences, a portion of human chromosome 21, aportion of the human X chromosome, and a portion of the human Ychromosome.
 3. The method of claim 1, wherein linking first and seconduniversal DNA segments into a first molecule in a linking reactiondependent on the first target nucleic acid comprises performing areaction selected from: (a) a PCR reaction, the reaction comprising: afirst primer having a 3′ portion that anneals to a first portion of thefirst target nucleic acid and a 5′ portion comprising the firstuniversal DNA segment; and a second primer having a 3′ portion thatanneals to a second portion of the first target nucleic acid and a 5′portion comprising the second universal DNA segment; whereinamplification of the first target nucleic acid using the first andsecond primers links the first and second universal DNA segments into asingle molecule; (b) a ligation reaction, wherein oligonucleotidescomplementary to the first target nucleic acid comprise 5′ and 3′ endsextending beyond the first target nucleic acid, and wherein theoligonucleotides serve as a template to ligate the first and seconduniversal DNA segment to the first target nucleic acid; (c) a ligationreaction, wherein the first universal DNA segment is ligated to thesecond universal DNA segment on the first target nucleic acid, therebylinking the first and second universal DNA segments together into asingle molecule; (d) a reverse transcription reaction, the reactioncomprising: generating a first cDNA strand by extending a first primerhaving a 3′ portion that anneals to a first portion of the first targetnucleic acid and a 5′ portion comprising the first universal DNAsegment, wherein the target nucleic acid is an RNA; and generating asecond cDNA strand by extending a second primer having a 3′ portion thatanneals to a portion of the first cDNA and a 5′ portion comprising thesecond universal DNA segment; wherein synthesis of the first and secondcDNA strands links the first and second universal DNA segments into asingle molecule; or (e) a linear polymerase extension reaction, whereinthe first universal DNA segment is linked to the first target nucleicacid by a polymerase that extends on the template complementary to thefirst universal DNA segment, and wherein a primer with a 5′ tailcomprising the second universal DNA segment introduces the seconduniversal DNA segment, thereby linking the first and second universalsegments into a single molecule.
 4. The method of claim 1, wherein step(a) is multiplexed such that multiple target nucleic acids of differentnucleic acid sequences are tagged with the same or different pairs ofuniversal DNA segments.
 5. The method of claim 1, wherein step (a)further comprises linking one or more additional pairs of universal DNAsegments into one or more additional molecules in a linking reactionspecific to one or more additional target nucleic acids, therebyproviding one or more additional tagged target nucleic acids in a singlereaction volume.
 6. The method of claim 1, wherein step (a) furthercomprises linking third and fourth universal DNA segments into a secondmolecule in a linking reaction specific to a second target nucleic acid,thereby providing a second tagged target nucleic acid.
 7. The method ofclaim 6, wherein the second tagged target nucleic acid is PCR amplifiedusing universal primers specific to the third and fourth universal DNAsegments, and wherein amplicons generated from amplification of thesecond tagged target nucleic acid form hairpin stems or circles thatresult in a second detectable signal that is distinguishable from thefirst detectable signal.
 8. The method of claim 1, wherein step (a)occurs in a first reaction location and step (b) occurs in one or moredifferent reaction locations.
 9. The method of claim 8, wherein the oneor more reaction locations of step (b) comprise a well, a nano-well, adroplet, an array structure, a flat surface or a bead surface.
 10. Themethod of claim 9, wherein the one or more reaction locations comprisean array structure or a bead surface, and wherein the first universalprimer is attached to the array structure, the flat surface or the beadsurface.
 11. The method of claim 8, wherein between steps (a) and (b), areaction mixture comprising the product of the linking reaction at thefirst location is transferred to the one or more different locations,wherein the reaction mixture is optionally diluted prior to, or during,transfer.
 12. The method of claim 11, wherein the first and seconduniversal primers are delivered to the one or more different reactionlocations, and wherein the first and second universal primers aredelivered prior to, during, or after the linking reaction mixture istransferred to the one or more different reaction locations.
 13. Themethod of claim 1, wherein the first universal primer comprises a firstlabel proximal to one end of the 5′ portion of the first universalprimer.
 14. The method of claim 13, wherein the first label comprises afluorescent dye.
 15. The method of claim 13, wherein the first universalprimer comprises a label quencher or FRET dye disposed proximal to thesame end or proximal to an end opposite the 5′ portion of the firstuniversal primer as compared to the first label, and wherein the labelquencher or FRET dye is disposed at an effective quenching or FRETdistance from the first label.
 16. The method of claim 15, wherein thefirst detectable signal resulting from hairpin stem or circle formationcomprises a change of FRET caused by changing the distance between thefirst label and the label quencher or FRET dye.
 17. The method of claim16, wherein changing the distance between the first label and the labelquencher or FRET dye is performed by a mechanism selected from: (a)removal of the label and/or label quencher or FRET dye from the ampliconor primer by a nuclease; (b) increasing the distance between the firstlabel and the label quencher or FRET dye, wherein the label quencher orFRET dye is initially disposed at an effective quenching or FRETdistance from the first label via a double-stranded hairpin stem or arandom coil ssDNA structure in the first universal primer, and whereinthe distance between the label quencher or FRET dye and the first labelis increased by the intramolecular hairpin that is formed within theamplicon generated upon extension of the first universal primer; (c)melting of an oligonucleotide from the first universal primer, whereinthe oligonucleotide comprises the label quencher or FRET dye, whereinthe first universal primer comprises the first label, and wherein thehairpin stem or circle generated upon extension of the first universalprimer melts the oligonucleotide from the first universal primer,thereby increasing the distance between the label quencher or FRET dyeand the first label; (d) forming a hairpin stem or circle disposes thefirst label at an effective FRET distance from a quencher or FRET dyeattached to an oligonucleotide that is complementary to the amplicongenerated upon extension of the first universal primer.
 18. The methodof claim 17, wherein the first label is disposed between the 3′ and 5′portions of the first universal primer, wherein the label quencher orFRET dye are disposed at the 5′ portion of the first universal primer ascompared to the first label, wherein the label quencher is removed fromthe amplicon with the nuclease, and wherein the labeled amplicon isdetected by capillary electrophoresis or hybridization to asurface-bound oligonucleotide comprising a region complementary to theamplicon.
 19. The method of claim 15, wherein the label quencher or FRETdye is disposed at a position on the first universal primer selectedfrom: a position between the 5′ and 3′ portions of the first universalprimer, and a position 5′ of the junction between the 5′ and 3′ portionsof the first universal primer.
 20. The method of claim 1, wherein thefirst universal primer comprises a polymerase blocking unit disposedbetween the 5′ and 3′ portions of the first universal primer.
 21. Themethod of claim 1, wherein the different pairs of universal primers arelabeled with different dyes, the different dyes having differentemission wavelengths, and wherein during step (b), the different dyeshaving different emission wavelengths are detected separately.
 22. Themethod of claim 4, wherein the target nucleic acids of different nucleicacid sequences are tagged with the same pair of universal DNA segments.23. The method of claim 16, wherein the first detectable signal ismeasured at every PCR cycle (real-time PCR).
 24. The method of claim 16,wherein the first detectable signal is detected as an end pointsubsequent to PCR.
 25. The method of claim 9, wherein the firstdetectable signal is detected as an end-point subsequent to PCR, andwherein the method further comprises counting the number of wells,nano-wells, droplets, DNA array features, or beads from which thedetectable signal is emitted.
 26. The method of claim 4, wherein themultiple target nucleic acids are selected from: a DNA, an RNA, aprimate nucleic acid, a rodent nucleic acid, a viral nucleic acid, abacterial nucleic acid, an archaea nucleic acid, a cDNA, a bisulphitetreated DNA, a cDNA corresponding to a short RNA, a genetic variant, amutation or insertion that confers drug resistance, a somatic mutation,a polymorphism, a single nucleotide polymorphism, a rare allele, aportion of the KRAS gene, a nucleic acid that exhibits a variation incopy number, an intron, an exon, an intron-exon boundary, a splicejunction, one or more dinucleotides corresponding to one or moremethylated or unmethylated CpG dinucleotides, a nucleic acid comprisingone or more restriction enzyme recognition sequences, a portion of humanchromosome 21, a portion of the human X chromosome, and a portion of thehuman Y chromosome, and wherein the presence and/or quantity of themultiple target nucleic acids is detected and combined into a diagnosticoutput.
 27. The method of claim 26, wherein the diagnostic output isdiagnostic for fetal aneuploidy, fetal sex, and/or fetal copy numbervariation, and wherein the fetal aneuploidy, fetal sex, and/or fetalcopy number variation is detected by digitally counting nucleic acidtargets indicative of fetal aneuploidy, fetal sex, and/or fetal copynumber variation from a maternal blood sample.
 28. The method of claim27, wherein the nucleic acid targets indicative of fetal aneuploidy,fetal sex, and/or fetal copy number variation are detected using a firstdetectable label, and control nucleic acid targets are detected using asecond detectable label, wherein the ratio of digital counts of thelabels is used to detect fetal aneuploidy, fetal sex, and/or fetal copynumber variation.
 29. The method of claim 15, wherein the firstuniversal primer is attached to a surface, wherein the label is disposedbetween the 5′ and 3′ portions of the first universal primer, whereinthe label quencher or FRET dye is disposed at a 5′ position relative tothe label, wherein the first or second universal primer is extended by apolymerase wherein the 5′ universal segment with label quencher or FRETdye hybridizes to the amplicon, and wherein extension of the universalprimer removes the quencher or FRET dye from the surface-bound universalsegment, thereby resulting or causing a change in the first detectablesignal on the surface.
 30. The method of claim 29, wherein the seconduniversal primer is in solution or attached to the surface, andoptionally the first universal primer comprises two oligos attached tothe surface in sufficiently high density such that 3′ and 5′ solutionends of the two oligos are sufficiently spatially close to each other toanneal to the same first molecule.
 31. The method of claim 8, whereinthe one or more different reaction locations of step (b) are disposedwithin or upon a detection plate or fluidic device, and wherein thefirst and second universal primers are preloaded into the one or moredifferent reaction locations.
 32. The method of claim 4, wherein poolsof encoding primers are inventoried or made to order, wherein theencoding primers are capable of amplifying a nucleic acid targetselected from a gene, a portion of a gene, a single nucleotidepolymorphism, a nucleic acid target that permits detection of a copynumber variation, a methylation target, an miRNA, and a somaticmutation, and wherein a combination of one or more pools of the encodingprimers is provided.
 33. The method of claim 32, wherein the combinationof one or more primer pools comprises a first pool of primers encoding aset of targets that optionally includes somatic mutations, singlenucleotide polymorphisms, copy number variants, and/or methylationtargets wherein the combination of one or more primer pools furthercomprises a second pool of encoding primers capable of amplifying one ormore cDNAs or miRNAs to determine gene expression levels.
 34. The methodof claim 33, wherein both pools are used on the same biological sample,and wherein the results of both pools can be combined and analyzedtogether.
 35. The method of claim 21, wherein the first and seconduniversal primers comprise fluorescent labels having different emissionwavelengths, wherein the first universal primer comprises a 5′ portionthat is substantially identical to a first strand of an internal portionof the tagged target nucleic acid, wherein the second universal primercomprises a 5′ portion that is substantially identical to thecomplementary strand of the internal portion of the tagged targetnucleic acid, wherein the 5′ portion of the first universal primer andthe 5′ portion of the second universal primer are sufficiently similarto the first strand and the complementary strand of the tagged targetnucleic acid, respectively, to anneal to the complement under PCRreaction conditions, and wherein amplification of both strands of thetagged target nucleic acid is measured independently using two or morecolors.
 36. The method of claim 35, wherein the target nucleic acidcomprises two closely spaced polymorphisms or methylation sites, andwherein the two closely spaced polymorphisms or methylation sites arehaplotyped using four colors.